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查看更多产品信息 Neon™ Transfection System 100 μL Kit - FAQs (MPK10025)
179 个常见问题解答
使用Neon设备后细胞存活率较低的可能原因包括：
1. 电学参数未优化彻底
2. 质粒品质不佳，如存在内毒素污染
3. 所制备质粒中盐份较高
4. 质粒用量过多
5. 细胞应激或受损
6. 多次使用同一Neon枪头
7. 枪头中存在微小气泡，从而导致电弧作用
使用Neon设备所获得转染效率较低的可能原因包括：
1. 电学参数未优化彻底
2. 所制备质粒中盐份较高
3. 质粒大于10 kb
4. 质粒浓度过低
5. 细胞应激，受损，或被支原体污染
6. 细胞密度过低或过高
7. 细胞传代次数过多
如需测定siRNA的Neon转染效率，我们推荐使用荧光标记的阴性对照siRNA来转染细胞（BLOCK-iT荧光寡聚物，目录编号13750062），并通过活细胞中荧光标记细胞的百分比来确定转染效率。不过，使用此方法时请牢记：通过荧光标记的阴性对照siRNA所确定的转染效率有高估的可能，这是因为通过显微镜进行的荧光检测无法区分进入胞内的siRNA和粘附在胞膜上的siRNA。如希望更为准确地测定转染效率，则需使用阳性对照siRNA（如靶向看家基因的siRNA）转染细胞，再检测靶向RNA或蛋白的敲低效果。
细胞存活率是指总细胞群体中确定具有活力的细胞数目。转染效率是指在所有存活细胞中成功表达您所构建载体的细胞数目（即，GFP阳性细胞）。
细胞活力也可通过碘化丙啶染色或台盼蓝拒染法来进行测定。对于贴壁细胞，可在染色前使用胰酶或TrypLE Express试剂进行细胞消化处理。用户可通过荧光显微镜选择适于检测GFP（发射波长：509 nm）的滤镜组来测定转染效率。可通过FACS或Countess自动细胞计数仪来进行细胞计数。
各类mRNA及其蛋白产物的稳定性和半衰期之间差异很大，所以根据经验来确定靶标敲低效果的最佳评估时间点至关重要。举例来说，据报道在哺乳动物细胞中，mRNA的半衰期在数分钟至数天的范围（Ross J, 1995, Microbiol Rev 59:423–450），而蛋白产物的半衰期短至数分钟，长至数天。通常情况下，敲低靶标mRNA的推荐时程在12-72小时，充分敲低靶标蛋白的时程在24-96小时。我们推荐用户在电转后8，24,48,72和96小时的时间点通过qPCR技术检测mRNA敲低效果，以确定最大敲低效率的时间点。同样地，用户也可通过ELISA（更精确）或免疫印迹（精确性稍低）技术进行时程分析以确定蛋白敲低效率。
最佳的蛋白分析时间点与所表达蛋白的稳定性有关。蛋白产物的半衰期可能短至数分钟，长至数天。对于半衰期短的蛋白（如荧光素酶），电转6-18小时后即可进行蛋白分析。对于GFP等更为稳定的蛋白，电转后24小时乃至稍晚一些均可进行分析。
为 10μL枪头优化的电转参数对于100 μL枪头也是适用的，但与缩放的任何变化一样，可能需要一些优化。在保持相同的细胞密度的同时，我们建议对电压设置进行微调，以达到最佳的转染效率。通常情况下，没有必要使用100 μL枪头进行24孔优化。
与任何电穿孔方法一样，电流被用来在细胞膜上打孔，从而导致膜蛋白的损伤。为了最大限度地恢复电穿孔后的膜损伤，使用适合特定细胞类型的培养基，并避免使用抗生素。根据细胞类型和蛋白质的不同，恢复时间可能不同。在电穿孔后不再扩增的原代细胞中，这种膜损伤可能是永久性的，从而阻碍了某些膜蛋白的恢复。首先尝试使用脂质体转染(见转染选择指南： https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).
电转完成后，请等待4-6小时再向细胞中加入抗生素。这是为了确保细胞膜的完整性彻底恢复以及贴壁细胞已完全贴壁。
按照Neon指南中的说明，您可在100 μL的系统中转染多至5百万个细胞。不过这个数据会根据不同类型的细胞大小而变化。
10 μL枪头可能使用的细胞少至1万个，但这可能取决于不同细胞类型的大小差异。我们的一位客户报告成功转染了5000个原代毛囊细胞。一般来说，尽量避免使用低密度的细胞，因为这可能会降低电穿孔的细胞活率。如果难以避免低细胞密度(如原代细胞)，调整电压以优化提高细胞活率。最佳的电穿孔条件与细胞类型有关。避免在培养基中使用抗生素，使用适合你细胞类型的培养基。
是的，使用Neon转染系统共转染质粒和siRNA是可行的，但可能需要做一些优化。
我们当前没有支持此项操作的数据，不过共转不同种质粒的操作是可行的。不过，DNA的用量需仔细斟酌，如果细胞中质粒DNA过载，或使用不恰当的质粒比例可能会导致细胞毒性的出现。因此，我们推荐您以低量DNA开始尝试共转实验的优化，并逐步增加DNA用量。如果观察到细胞毒性，则需尝试多种不同的质粒比例。
Neon细胞数据库包含多种常用细胞类型的优化转染方案。由于您所用细胞系的传代次数和/或培养条件，或分离步骤不会与我们所用的完全一致，因此您可基于特定细胞系对这些实验条件进行微调。数据库中的实验条件可作为您自身优化工作的起点。对于我们数据库中未列举的细胞系，Neon设备中内置了预先编程的优化实验方案。
在大多数情况下，针对某一细胞系或原代细胞的质粒转染而进行优化的仪器设置也适用于siRNA。不过，这些设置参数并不一定是递送siRNA的最佳参数。因此，如需提升靶标的敲低效率，可能仍需要额外的优化操作。对于未经质粒DNA转染条件优化的细胞系或原代细胞，一组24孔优化实验可能是找到优化条件的最佳手段。请记得在每个测试条件中加入一个siRNA阴性转染对照，以便对敲低效率进行标准化计算。
一个比较好的尝试起点是同种细胞类型的质粒电转参数。Neon细胞数据库（https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html）为多种常用类型的细胞准备了优化的质粒转染方案。如果Neon细胞数据库未包含目的细胞类型，您可使用Neon设备中预先载入的24孔优化实验方案。如果您需要进一步的帮助，请联系技术支持（LifeScience-CNTS@thermofisher.com）。
Neon转染所用siRNA浓度指的是培养基中浓度，而非Neon枪头中电转体系的siRNA浓度。举例来说，如果使用100 μL Neon枪头对培养在24孔板，500 μL培养基中的细胞进行转染操作，则siRNA浓度需按照500 μL培养基进行测量，而非按照100 μL电转体系的体积进行测量。
可以。Neon转染系统适用于任何RNAi介质（siRNA，shRNA，miRNA）。您可应用与DNA相同的细胞特异型实验方案，或利用预先编程的24孔板优化条件进行实验方案优化。
如果您使用同种质粒/siRNA和同种细胞，电解缓冲液最多可使用10次，之后需一并更换反应管和缓冲液。如果您使用不同的质粒/siRNA或细胞类型，我们推荐您使用两次即更换缓冲液，以避免残留污染。
我们强烈建议不要洗涤Neon枪头。洗涤无法去除枪头上面粘附的DNA或siRNA，还会增加样本间交叉污染的风险。另外，枪头也无法消毒，也很容易增加培养物被微生物污染的风险。
为了避免样本间的残留污染，我们不推荐您重复利用Neon枪头。不过如果您需要执行24孔优化操作或重复操作两组转染实验，这时的枪头可使用两次。我们之所以这样推荐的原因是枪头中的电极表面镀有24k金，每次施加电脉冲时都会释放掉一些金离子。因此，重复使用枪头会使金包被层变薄，从而导致枪头的导电性发生改变。我们发现在使用三次后的枪头上这种变化即可测量到。如果您希望向细胞施加绝对准确的电压和电流，每个枪头您只能使用两次。
Neon转染管是一次性用品，我们推荐您最多使用每管转染10次（相同的质粒/siRNA/细胞类型），以最大限度地减少交叉污染的可能性。此外，我们强烈推荐您使用一个全新的Neon管来转染不同类型的DNA/siRNA或细胞，以避免交叉污染。如果您需要为实验匹配额外的Neon管，可单独进行购买（目录编号 MPT100）。
我们目前尚未提供此类服务。Neon转染系统专为提升转染条件的优化效果而设计。通常情况下，三轮优化就足以为任意种类的细胞系或原代细胞取得最佳的仪器设置参数。除非您以极少量组织或难于处理的组织制备细胞，否则条件优化工作的时长不会超过一周，并且在花费上远远低于定制服务。
这些设置是基于我们对大量细胞系和原代细胞的内部经验来进行挑选的。如果所有这些参数设置都无法成功帮助您将质粒DNA转染进细胞，那么其他条件也未见得能成功。不过，如果使用某些设置获得的转染效率较低，您可对电压、脉冲宽度和脉冲次数等参数进行精细调节来优化条件，则转染效率很可能得到进一步提升。
如果您在Neon细胞数据库（https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html）中找到一种与您所用细胞具有相同组织来源的类似细胞株时，您可借用这一细胞的参数。这并不保证您能够获得最佳结果，不过是一个好的尝试起点。举例来说，如果您拥有293T细胞，而又在Neon细胞数据库中找到了HEK293细胞的转染方案，您就可将HEK293细胞的电转参数用于293T细胞，因为两者都源自人胚肾。
您可在Neon设备中使用预先编程好的24孔优化方案来优化您的细胞转染条件。
请联系技术支持 来进一步商讨。
强电场的应用会削弱细胞膜结构，导致膜孔的形成，抗生素也会由此进入细胞内部。后续产生的毒性代谢中间产物很可能介导细胞死亡的发生。此外，链霉素会与真核细胞核糖体相结合，进而直接干扰蛋白翻译过程。如果您的细胞需要培养于含抗生素的条件下，您可在电转操作数小时之后再加入抗生素。
某些大质粒上所观察到的毒性效应与其长度并不相关，而更可能是由质粒所含序列，质粒制备过程引入的LPS一类污染物，电转极大量的质粒等因素所致。请保持使用基于阴离子交换层析的试剂盒（如我们的PureLink HiPure， PureLink HiPure Expi 或 PureLink Expi Endotoxin-Free 质粒纯化试剂盒）来制备转染级的质粒DNA，同时避免柱体超载——否则可能会产生低纯度的质粒。
通常情况下，电转染是一种质粒大小依赖的转染技术，转染效率将随质粒大小的增加而降低。我们在内部实验室中通常使用4-7kb的质粒，而转染20kb以内长度的质粒应该都不会有问题。使用超过这一长度的质粒将很可能导致转染效率变低。初步研究结果提示细菌人工染色体（BAC）也能够通过本系统进行转染，只是转染效率较低。请留意，对摩尔数来说，一个6kb长度质粒的1mg对应于12kb质粒的2mg。这就是为什么在比较不同长度质粒的转染效率时要将此因素考虑进来的原因。举例来说，如果需要将1mg 10kb的质粒与150kb的BAC的转染效率做比较的话，就需要使用15mg的BAC。但这是不可行的，因为如此大量的DNA对细胞是有毒的。另一方面，这并不意味着Neon无法转染BAC。只是DNA使用量太大时转染效率会很低。
不含特殊重组序列的环状与线性化质粒的转染能力相同，也按照相近的概率整合进入基因组。不过，质粒上的重组位点会受线性化处理的影响，自由末端的整合效率会高于中间连续序列。在大多数情况下，您可通过质粒线性化决定质粒上重组发生的位置，从而保护表达组件。
我们没有内部实验数据，不过来自客户的一些报告提示本系统能够在多种细胞系上完成这一操作。
我们推荐您使用阴离子交换层析法来制备转染级的质粒DNA。我们的PureLink HiPure质粒纯化试剂盒系列产品即使用了这一技术。对于大质粒的抽提（>50 kb），请勿使用含有过滤器或沉淀器的PureLink HiPure纯化试剂盒，以避免质粒受损。我们不推荐使用离心柱来纯化质粒，因为它们所含的硅胶膜去除杂质的能力无法达到阴离子交换树脂同一水平。对追DNA的大量制备，我们推荐使用 PureLink HiPure Expi 或 PureLink Expi Endotoxin-Free 质粒纯化试剂盒。
我们目前尚未为此操作提供经过验证的方案。
Neon转染系统可用于电转衣藻。请参见GeneArt Chlamydomonas Protein Expression Vector (货号A24231)的说明书中推荐的电转方案。目前，我们尚未提供细菌电转的Neon实验方案。
使用Neon系统进行电转的过程中，某些有聚集趋势的细胞（如PC-12细胞）可能“偶尔”出现细胞融合的副效应，不过很可惜，我们并未专门提供Neon程序来优化细胞融合条件。
虽然我们的Neon转染试剂盒中不含对照质粒，但pJTI R4 Exp CMV EmGFP pA Vector (Cat. No. A14146)可用来做为瞬时表达对照。
有的。您可在此处（https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-citation.html）访问Neon引用列表。
在我们的Neon细胞数据库（https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html）中，我们为每一种细胞类型提供了基于通用型电解缓冲液优化的验证过的电转参数。由于您使用细胞的传代次数和/或培养条件，或分离步骤不会与我们所用的完全一致，因此您可基于特定细胞系对这些实验条件进行微调。数据库中的实验条件可作为您自身优化工作的起点。对于我们数据库中未列举的细胞系，Neon设备中内置了预先编程的基于24孔板的优化实验方案。
对于日常清洁，我们推荐使用70%的乙醇来清洁Neon设备与Neon移液工作站的表面。不要使用强去垢剂或有机溶剂清洁设备组件。要进行深层清洁，请使用广谱的医用消毒剂。避免将任何液体内洒到Neon移液工作站内部。对于Neon移液工作站内的意外喷洒(如缓冲液、水、咖啡)，应从主设备上断开，用干燥的实验室用纸擦拭。倒置并在室温下放置24小时确保完全干燥。不要使用烤箱或高压锅来干燥Neon移液站。
Neon移液器已由生产商永久校准，用户无需进一步调校。
Neon转染枪头可单独购买（目录编号 MPT100）。其它任意组分的批量定制可通过客户定制服务完成，更多信息请联系我们的技术支持。
Neon试剂盒的保质期为从购买日起一年时间。
是的，Neon枪头可匹配Digital Bio/NanoEntech的移液器。
脉冲间隔固定在1ms。
Neon设备使用了方波脉冲；脉冲宽度为1-100ms，脉冲数量范围在1-10次。电压范围在500-2500V之间。
E2型缓冲液比E型缓冲液拥有更高的渗透压。这一更高的渗透压能够避免电转体系从100 μL Neon枪头中漏出，100 μL的 Neon枪头比10 μL Neon枪头的末端孔径更大（Neon枪头的孔直径：100 μL 枪头为2.10 mm；10 μL枪头为0.65 mm）。
我们不提供重悬缓冲液R的单品。但批量定制可通过客户定制服务完成。更多信息请联系我们的技术支持。
对于血液来源的原代悬浮细胞，如原代T细胞与B细胞，PBMC，单核细胞和骨髓来源细胞，我们推荐使用T缓冲液代替标准的R缓冲液。这些细胞比常见细胞系的体积更小，因此需要更高的电压来达到成功的电转效果。如果您使用R缓冲液并施加高电压（超过1800V），无论使用怎样的细胞数量和转染条件，您都会观察到电火花或电弧。R缓冲液可施加的最高电压为1900V左右。T缓冲液的成份区别于R缓冲液，具有更低的导电性，因此T缓冲液可胜任更高的电压条件。
R型与T型重悬缓冲液均适用于在电转操作前对细胞进行重悬操作。R型重悬缓冲液为标准的细胞重悬缓冲液，可用于重悬已建系的贴壁细胞，悬浮细胞，以及原代贴壁细胞。T型重悬缓冲液为细胞重悬缓冲液的一个备选，可用于重悬原代T细胞与B细胞、PBMC、单核细胞和骨髓来源细胞。其组成成份不同于R型缓冲液，由于其导电性较低，从而可应用更高电压。本品不适用于建立好的细胞系或已经培养了一段时间的原代细胞。如果用户无法即刻明确这两种缓冲系统哪种更适合自己的细胞，我们推荐您分别进行独立优化实验，以测试其实际使用效果。
重悬缓冲液（缓冲液R或T）适用于在电转前重悬细胞，而电解缓冲液（缓冲液E或E2）适用于电转过程，需要在电转前加入Neon管中。
Neon转染系统将DNA、mRNA、siRNA和蛋白质高效地传递到所有哺乳动物细胞类型中，是脂质体转染效率低时的最佳选择。对于免疫细胞或血源细胞，我们通常推荐Neon转染系统。对于神经元细胞，我们推荐使用Lipofectamine MessengerMAX转染试剂递送mRNA。对于所有的干细胞，我们推荐Lipofectamine Stem转染试剂。请访问我们的转染试剂选择指南：https://www.thermofisher.com/cn/zh/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html
慢病毒也非常有效地传递载体，用于所有细胞类型的基因表达和RNAi应用。我们建议使用LV-MAX慢病毒生产系统生产高滴度慢病毒颗粒。
不同于标准的电转杯容器，Neon系统使用了专利的生物相容性移液枪头作为电转容器。移液枪头中内置金包被电极丝的设计能够为细胞悬液提供更为均匀的电场和更低的pH梯度。因此，这一设计能够更好地维持生理条件，从而达到比传统电转法更高的细胞存活率（Kim JA, Cho K, Shin MS, et al.（2008）A novel electroporation method using a capillary and wire-type electrode.Biosens Bioelectron 23(9):1353–1360）。
Neon转染系统是由Digital Bio/NanoEntek公司生产的Microporator(MP-100)的第二代转染系统。这两种仪器都有一个独特的吸头式电极室，而不是一个标准的电转杯。这些仪器的性能相当，但是Neon转染系统有以下改进的特性：
- 改进的用户触摸屏界面，更大的显示区域
- 最新固件(可在仪器管理部分查看：https://www.thermofisher.com/cn/zh/home/products-and-services/services/instrument-qualification-services/instruments-and-services-portal.html)
- 预设一个24孔板优化程序
- 改进的传感器连接器设计
- >130种经验证的细胞特异的在线操作步骤
我们不携带适配器，以允许前一个设备和当前设备之间的兼容性。这两个系统的试剂盒和组件完全相同，只是在Neon转染系统中被标记为Neon。旧说明书可用于Neon电转仪。更多关于Neon转染系统历史的信息可以在这里找到：https://www.thermofisher.com/cn/zh/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/history-of-the-neon-transfection-system.html.
这里列举了一些Neon转染系统的特性：
1. 移液管顶端腔室设计，易于细胞处理，电场均匀，由于在电穿孔过程中pH值变化极小，具有较高的存活率。
2.Neon转染系统使用了一款通用型的转染试剂盒（Neon试剂盒），能够兼容包括原代细胞和干细胞在内的多种哺乳动物细胞类型，进而能够帮助用户免除分别为每一类细胞优化缓冲体系的麻烦。本品提供了两种细胞重悬缓冲液，能够覆盖各类细胞应用。T缓冲液适用于原代T细胞与B细胞、PBMC、单核细胞与骨髓来源细胞，而R缓冲液适用于已建系的贴壁细胞和悬浮细胞，以及原代贴壁细胞。
3. Neon转染系统能够广泛兼容不同数量的细胞样本。如果使用100 μL转染体系，最多可转染5 × 106 个细胞；如果使用10 μL转染体系，最少可转染1× 104个细胞。
4. 提供超过130个已验证的在线操作步骤，通过优化使其具备了易用性和简单性。访问我们的Neon 细胞数据库(https://www.thermofisher.com/cn/zh/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html)
5. 预制了24孔板优化方案，以便快速根据细胞类型确定最佳的电穿孔参数。
6. 灵活的负载和应用。可导入DNA, mRNA, siRNA和蛋白质。
是的。我们推荐您以琼脂糖凝胶来检验DNA的完整性，观察其是否发生降解。超螺旋的质粒会比线性质粒泳动得更快。带切口的质粒将比线性质粒泳动得更慢。您所制备DNA产物中的“带切口”DNA成份应低于20%。更高比例的带切口DNA会导致转染效率的显著下降。
是的。为了检查您的DNA质量，我们强烈建议确认A260:A280和A260/230的比值都在1.6-2.0之间，并通过琼脂糖凝胶电泳检查DNA降解情况。
使用Neon转染系统进行大质粒转染的建议如下:
-制备高浓度质粒(如5 mg/mL)以使DNA体积小于总电转反应体系的10%。
-使用高纯质粒来避免细胞毒性问题。确认A260/280和A260/230的比率在1.6-2.0之间。对于无内毒素质粒DNA，我们推荐使用PureLink Expi质粒纯化试剂盒
-确保DNA的量是在产品手册中为10或100 ul枪头建议的范围内。根据细胞大小、细胞密度和质粒的变化，有些优化是正常的。
-琼脂糖凝胶电泳验证DNA完整性和无降解。
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不会，这些沉淀是不可逆的。如果该Neon试剂盒仍在1年有效期内，请联系技术支持部门进行更换。
如需测定siRNA的Neon转染效率，我们推荐使用荧光标记的阴性对照siRNA来转染细胞（BLOCK-iT荧光寡聚物，货号13750062），并通过检测活细胞中荧光染色细胞的百分比来确定转染效率。不过，在使用此方法时请切记：通过荧光标记的阴性对照siRNA所确定的转染效率有高估转染效率的可能，这是因为通过显微镜进行的荧光检测无法区分进入胞内的siRNA和粘附在胞膜上的siRNA。如需更为准确地测定转染效率，需要使用阳性对照siRNA（如靶向看家基因的siRNA）转染细胞，再检测靶标RNA或蛋白的敲低效果即可。
这一现象有几种可能的原因：单核细胞与巨噬细胞会响应极低水平的内毒素（LPS），而后者可能由您的质粒DNA引入的。请确保您使用的质粒DNA经过例如PureLink HiPure，PureLink Expi，或Purelink Expi Endotoxin-Free质粒纯化试剂盒一类的阴离子交换层析法的纯化。如果您仍然观察到激活现象，您可能需要对质粒进行第二轮的阴离子交换层析纯化。如果您仍无法避免细胞激活的现象，则质粒中可能包含了刺激gamma干扰素合成的序列。也可能您的培养基（包括本批FBS）中存在某些成份导致了激活效应。请确保没有这些成份激活细胞。分离单核细胞的步骤也同样很重要。我们推荐阴性筛选而非阳性筛选，因为这样的筛选过程能够让单核细胞不“碰触”抗体。我们的电转缓冲液能够确保不含内毒素成份，因此不会导致单核细胞/巨噬细胞的激活。
电弧可能是由高电压或脉冲长度设置，DNA样本中的高盐或污染物，不正确的细胞密度，以及在细胞样本混合过程中形成的气泡引起的。我们建议进行24孔板优化(参见产品手册)以确定适合您的细胞类型的最佳电穿孔参数。请确保质粒DNA的A260/280和A260/230的比率在1.6-2.0之间。使用血球计或Countess II自动细胞计数仪精确测定细胞密度。轻轻混合样品以避免气泡形成。吸取样本时要缓慢平稳以避免吸入空气。
Here are possible causes for low transfection efficiency using the Neon device:
1. Sub-optimal electrical parameters
2.Poor plasmid quality such as endotoxin contamination
3 .Plasmid preparation containing high salt
4. Plasmid quantity too high
5. Cells are stressed or damaged
6. Using same Neon tip more than two times
7. Microbubbles in tip, causing arcing
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Here are possible causes for low transfection efficiency using the Neon device:
1. Sub-optimal electrical parameters
2. Plasmid preparation containing high salt
3. Plasmid larger than 10 kb
4. Plasmid concentration too low
5. Cells are stressed, damaged, or contaminated by Mycoplasma
6.Cell density too low or too high
7. Cells with high passage number
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
To determine the Neon transfection efficiency for siRNA, we recommend transfecting the cells with a fluorescent-labeled negative control siRNA (BLOCK-iT Fluorescent Oligo, Cat. No. 13750062) and measuring the transfection efficiency by the percentage of fluorescent-stained cells among viable cells. However, keep in mind that there is a caveat with this approach: the transfection efficiency determined by fluorescent-labeled negative control siRNA may over-estimate the transfection efficiency, as fluorescence detection with a microscope does not distinguish the siRNA that enters the cell from the siRNA that sticks to the cell membrane. To measure transfection efficiency more accurately, one needs to transfect the cells with a positive control siRNA such as the one that targets a house-keeping gene, and measure the knockdown of target RNA or protein.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Cell viability is the number of cells that are confirmed viable from a total cell population. Transfection efficiency is the number of cells that are successfully expressing your construct out of the total number of viable cells (i.e., GFP-positive cells).
Cell viability can be determined by staining cells with propidium iodide or by the trypan blue exclusion method. For adherent cells, cell detachment can be performed using Trypsin or TrypLE Express enzyme prior to staining. Transfection efficiency can be determined using a fluorescence microscope with filter settings appropriate for the detection of GFP (emission: 509 nm). Cells may be counted either by FACS or using the Countess Automated Cell Counter.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
As the stability and half-life of various mRNAs and their protein products varies, it is important to empirically determine the best time points for assessing target knockdown. For example, it has been documented that in mammalian cells, mRNA half-life can range from minutes to days (Ross J, 1995, Microbiol Rev 59:423–450) while the half-life of protein products can range from less than a few minutes to several days. In general, the recommended time course ranges from 12 to 72 hours to knock down target mRNA and 24 to 96 hours to adequately knock down target proteins. We recommend measuring mRNA knockdown by qPCR at 8, 24, 48, 72, and 96 hours post-electroporation to determine the time point for maximum knockdown. Also, perform time-course analysis to determine protein knockdown by ELISA (more accurate) or immunoblotting (less accurate).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
The optimal time point for analysis of protein expression is related to the stability of the protein being expressed. The half-life of protein products can range from less than a few minutes to several days. For a short-lived protein (like luciferase), protein expression analysis should be done at 6-18 hours post-electroporation. For a more stable protein such as GFP, the analysis can be done 24 hours post-electroporation or even a little later.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Electroporation parameters optimized for the 10 µL tip may be used for the 100 µL Neon tip, but as with any changes in scaling, some optimization may be required. While keeping to the same cell density, we recommend fine-tuned adjustments of the voltage settings to achieve optimal transfection efficiency. A 24-well optimization with the 100 µL tips is usually not necessary.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
As with any electroporation method, electrical current is used to create pores in the cell membrane, resulting in some membrane protein damage. To maximize recovery after electroporation, use medium that is optimized for the specific cell type and avoid antibiotics. The recovery time may vary and will depend on cell type and protein. In primary cells, which do not proliferate after electroporation, this membrane damage could be permanent so that it hinders certain membrane protein recovery. Try using lipid transfection first (see Transfection Selection Guide; https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
After electroporation, wait for about 4-6 hours before adding antibiotics back to the cells. This is to make sure that the membrane integrity has been restored and to allow adherent cells to attach to the culture vessel.
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According to Neon guidelines, you can run up to 5 million cells in 100 µL, this number may vary depending on the size of the cell type.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
As few as 10,000 cells may be used with the 10 µL tips, but this may vary depending on size differences among cell types. One of our customers reported successful transfection of 5,000 primary hair follicle cells. As a general rule, try to avoid using low cell densities as this could reduce viability during electroporation. If it is difficult to avoid low cell densities (ex. primary cells), adjust the voltage to optimize for improved viability. Optimal electroporation conditions are cell type-dependent. Avoid antibiotics in the medium and use medium appropriate for your cell type.
For helpful cell-specific electroporation conditions, please visit our Neon cell-specific protocols database https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html?SID=fr-neon-3
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Yes, you may co-transfect both plasmid and siRNA together at the same time but some optimization may be necessary.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
We currently do not have data to support this, but co-transfection of different plasmids should work. However, the amount of DNA should be carefully titrated, since overloading the cells with plasmid DNA or using unfavorable ratios of the plasmids may cause toxicity. Therefore, we recommend starting optimizations of co-transfection experiments with low amounts of DNA followed by a stepwise increase. Various ratios of the plasmids should be tested if toxicity is observed.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
The Neon Cell Database (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html) offers optimized protocols for many commonly used cell types. However, these conditions may have to be modified slightly for your particular cell line, since passage number and/or culture conditions or cell isolation procedures may not be the same as ours. The conditions listed should be understood as a starting point for your own optimization. For cell lines that are not listed in our database, there is a pre-programmed 24-well optimization protocol built into the Neon device.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
In most cases, instrument settings that were optimized for a certain cell line or primary cell type using a plasmid will also work with siRNA. However, those settings may not be the most optimal ones for the delivery of siRNA. Therefore, additional optimization may be required to improve knockdown efficiency of the target. For cell lines or primary cell types that have not been optimized with plasmid DNA, a 24-well optimization is the best approach to find optimal conditions. Keep in mind that for every condition tested, a negative control siRNA needs to be transfected in order to normalize knockdown efficiency.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
A good start is to use the plasmid electroporation parameters for the same cell type. The Neon Cell Database (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html) contains optimized plasmid electroporation parameters for many commonly used cell types. If the Neon Cell Database does not contain your cell type of interest, you can use the 24-well optimization protocol that is pre-loaded on the Neon device. Please contact Technical Support at techsupport@thermofisher.com if you should need further assistance.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
The siRNA concentration in Neon transfection refers to the siRNA concentration in the culture medium and not to the siRNA concentration in the electroporation content in the Neon tip. For example, if electroporation is performed with the 100 µL Neon tip and the transfected cells are plated in a 24-well plate that contains 500 µL culture medium, the siRNA concentration is measured as the concentration in the 500 µL culture medium and not the concentration in the 100 µL electroporation content.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Yes. The Neon Transfection System can be used for any RNAi substrate (siRNA, shRNA, miRNA). You can use the same conditions described in the cell type-specific protocol for DNA or pre-programmed 24-step optimization protocol.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
If using the same plasmid/siRNA and the same cell type, one can use the Electrolytic Buffer for up to 10 times and then change the tube and buffer together. If a different plasmid/siRNA or cell type is used, we recommend changing the buffer after one usage to avoid carryover contamination.
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We strongly advise against washing the Neon tips. Washing will not remove DNA or siRNA attached to the tip and will increase the risk of cross-contaminating the samples. Also, the tips cannot be sterilized, easily increasing the risk of microbial contamination of cultures.
To avoid contamination caused by carryover from sample to sample, we recommend that you do not re-use the tip. However, if performing a 24-well optimization or transfections performed in duplicate, the tips may be used up to 2 times. The reason for this recommendation is that some of the gold coating the wire electrode inside the tip is released each time an electrical pulse is delivered. Therefore, repeated use of the tip will result in a thinning of the gold coating, causing the conductivity of the tip to change. We found that this effect becomes measurable after three uses. To ensure correct voltage and current are delivered for every electroporation, use the tip only twice.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
The Neon Transfection Tubes are disposable and we recommend using each tube for a maximum of 10 times for the same plasmid/siRNA/cell type, to minimize the possibility of cross-contamination. In addition, we strongly recommend that a new Neon tube be used for a different plasmid DNA/siRNA or cell type, to avoid cross-contamination. If you need extra Neon tubes to accommodate your experiment, they can be purchased separately (Cat. No. MPT100).
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
We do not offer such a service at this time. The Neon Transfection System is designed to facilitate the optimization of transfection conditions. Typically, three rounds of optimization are sufficient to find the best instrument settings for any given cell line or primary cell type. Unless you prepare your cells from little amounts of tissue or tissue that is difficult to process, optimizing the conditions should not take more than a week and would cost much less than a custom service would.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
These settings were selected based on our experience optimizing numerous cell lines and primary cells in-house. If none of these settings transfect plasmid DNA into your cells, it is unlikely that other conditions will. However, if low transfection efficiencies are obtained with some of the settings, it is likely that they can be further increased by performing additional optimizations to fine-tune your parameters for voltage, pulse width, and number of pulses.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
We recommend reviewing our Neon Transfection System Protocols and Cell Line Database (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html) to identify a cell type that is similar in tissue origin, to your own cell and try the recommended parameters in the protocol. This is a good starting point, but some optimization may be needed (ex. adjusting voltage). For example, if you have 293 T cells and you find a protocol for HEK293 cells in the Neon Cell Database, you can use the electroporation parameters of HEK293 cells for 293 T cells, since both are derived from human embryonic kidney.
- You can use the pre-programmed 24-well optimization protocol in the Neon device to optimize conditions for your cell type.
- Contact Technical Support at techsupport@thermofisher.com for further discussion.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Application of a strong electrical field weakens the cell membrane and induces pore formation, which allows the antibiotics to enter the cells. Cell death is induced most likely via toxic metabolic intermediates. In addition, streptomycin has been shown to bind to eukaryotic ribosomes and may directly interfere with protein translation. If your cells need to be cultured in the presence of antibiotics, you can add them back a few hours after electroporation.
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The cytotoxic effects observed with some large plasmids are often not related to their size, but are more likely due to sequence-specific effects, contaminants carried over during plasmid preparation (ex. LPS), or very large DNA amounts delivered during electroporation. Always use anion-exchange chromatography-based kits (such as our PureLink HiPure, PureLink HiPure Expi, or PureLink Expi Endotoxin-Free Plasmid Isolation Kits) to prepare transfection-grade plasmid DNA. During plasmid DNA isolation, avoid overloading the columns, as this will result in plasmid preparations of low purity.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
In general, electroporation is a size-dependent transfection technique and transfection efficiency declines as plasmid size increases. We routinely use plasmids of 4-7 kb in our laboratories, and plasmids up to approximately 20 kb should not be a problem. Using plasmids larger than this will most likely result in lower transfection efficiency. Preliminary results indicate that bacterial artificial chromosomes (BACs) can be transfected as well, but with a low transfection efficiency. Keep in mind that in terms of molarity, 1 mg of a 6 kb plasmid corresponds to 2 mg of a 12 kb plasmid. This is why plasmid size is taken into consideration when comparing transfection efficiencies between plasmids of different lengths. For example: when comparing the transfection efficiency between 1 mg of a 10 kb plasmid and the transfection efficiency of a 150 kb BAC, 15 mg of the BAC would have to be used. This is not feasible since DNA amounts that large will cause cytotoxicity. On the other hand, this does not mean that BACs cannot be transfected using the Neon system. However, transfection efficiencies with a large amount of DNA will be very low.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Circular and linearized plasmids (that do not contain special recombination sequences) transfect into the cell and integrate into the genome with similar efficiencies. However, the area of recombination on the plasmid can be influenced by linearization, as loose ends are preferred over continuous stretches of sequence. By linearizing the plasmid, you can determine the position within the plasmid where the recombination occurs, thereby conserving the expression cassette in most cases.
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We do not have in-house data, but several reports from customers using a variety of cell lines suggest that it works.
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We recommend using anion-exchange chromatography to prepare transfection-grade plasmid DNA. This technology is the basis of our PureLink HiPure line of plasmid purification kits. For large plasmids (>50 kb), do not use the PureLink HiPure purification kits that contain filters or precipitators to avoid damage to your plasmid. We do not recommend using spin columns for plasmid purification, as they contain silica membranes that do not remove impurities to the same extent as anion-exchange resins. For high-yield plasmid DNA isolation, we reccomend the PureLink HiPure Expi or the PureLink Expi Endotoxin-Free Plasmid Purification Kits.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Currently, we do not have a validated protocol.
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The Neon Transfection System may be used for electroporation of Chlamydomonas. Please refer to the GeneArt Chlamydomonas Protein Expression Vector (Cat. No. A24231) manual for Neon instructions. Currently, we do not offer a Neon protocol for electroporation of bacterial cells.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Cell fusion may occur "accidentally" as a side-effect during transfection using the Neon system, for some cell-types that tend to form cell clusters (e.g., PC-12 cells), but unfortunately, we do not offer a Neon program that is optimized for cell fusion applications.
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Although our Neon Transfection kits do not include a control plasmid, the pJTI R4 Exp CMV EmGFP pA Vector (Cat. No. A14146) may be used as a transient expression control.
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Yes. You may access the Neon citations here (http://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-citation.html/).
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For each cell type in our Neon Cell Database (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html), we offer validated electroporation parameters optimized with a universal electrolytic buffer. These conditions may have to be modified slightly for your particular cell line, since passage number and/or culture conditions or cell isolation procedures may not be the same as ours. The conditions listed should be understood as a starting point for your own optimization. For cell lines that are not listed in our database, there is a 24-well pre-programmed 24-well optimization protocol built into the Neon device.
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For routine cleaning, we recommend cleaning the surface of the Neon device and Neon Pipette Station with 70% ethanol. Do not use harsh detergents or organic solvents to clean the unit. For deeper cleaning, use a broad-range medical disinfectant. Avoid spilling any liquid inside of the Neon Pipette Station. For accidental spiils (e.g., buffer, water, coffee) inside the Neon Pipette Station, disconnect the station from the main device and wipe the station using dry laboratory paper. Invert and leave the station for 24 hours at room temperature for complete drying. Do not use an oven or autoclave to dry the Neon Pipette Station.
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The Neon Pipette is permanently calibrated by the manufacturer and does not require any further calibration.
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The Neon transfection tubes are available separately (Cat. No. MPT100).
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The shelf life of the Neon kits is 1 year from the date of shipment.
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Yes, the Neon tip fits into the Digital Bio/NanoEntech pipette.
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The interval between pulses is fixed at 1 ms.
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The Neon device uses a square pulse wave; the pulse width range is 1-100 ms and the pulse number range is 1-10. Volts range from 500-2500 V.
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The E2 buffer has higher osmolarity than E buffer. Higher osmolarity prevents the leakage of electroporation content from the 100 mL Neon tip, which has a aperture hole at the tip end than the 10 mL Neon tip (pore diameter of the Neon tips: 100 mL tip = 2.10 mm; 10 mL tip = 0.65 mm).
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Sorry, we do not offer Resuspension Buffer R as a stand-alone item.
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We recommend using T buffer instead of the standard R buffer for primary blood-derived suspension cells such as primary T and B cells, PBMCs, monocytes, and bone marrow-derived cells. These cells are smaller than regular cell types and require higher voltage for successful electroporation. If high voltage (>1800 V) is applied to buffer R, sparks or arcing may be observed, regardless of cell number and other conditions. The maximum voltage for R buffer is approximately 1900 V. Buffer T composition differs from that of Buffer R to allow the application of higher voltages due to lower conductivity.
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Both R and T Resuspension Buffers are used to resuspend cells prior to electroporation. Resuspension Buffer R is used for established adherent and suspension cells as well as primary adherent cells. Resuspension Buffer T is an alternative cell resuspension buffer for primary T and B cells, PBMCs, monocytes, and bone marrow-derived cells. Buffer T differens in composition from that of Buffer R and allows the application of higher voltages due to lower conductivity. It does not work with established cell lines or primary cells, which have been kept in culture for some time. In situations where it is not immediately clear whether Buffer R or Buffer T would work, we recommend testing both in separate optimization experiments.
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The Resuspension Buffer (Buffer R or T) is used to resuspend the cells prior to electroporation, whereas the Electrolytic Buffer (Buffer E or E2) is used for electroporation and is added to the Neon tube prior to electroporation.
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The Neon Transfection System delivers DNA, mRNA, siRNA, and proteins efficiently into all mammalian cell types and is the best option when lipid-based transfection efficiencies are low. For immune or blood-derived cells, we generally recommend the Neon Transfection System. For neuronal cells, we recommend mRNA delivery with Lipofectamine MessengerMAX Transfection Reagent. For all stem cells, we recommend Lipofectamine Stem Transfection Reagent. Please visit our Transfection Reagent Selection Guide:
https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html
Lentiviruses are also very efficient in the delivery of vectors for all cell types for gene expression and RNAi applications. We recommend producing high titer lentivirus particles using our LV-MAX Lentiviral Production System.
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Unlike standard cuvette-based electroporation chambers, the Neon system uses a patented biologically compatible pipette tip chamber. The design of a gold-coated wire electrode inside a pipette tip has been shown to produce a more uniform electrical field and a lower pH gradient across the cell suspension. Therefore, this design allows for better maintenance of physiological conditions, resulting in very high cell survival compared to conventional electroporation (Kim JA, Cho K, Shin MS, et al. (2008) A novel electroporation method using a capillary and wire-type electrode. Biosens Bioelectron 23(9):1353–1360).
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The Neon Transfection system is the second generation of the Microporator (MP-100) from Digital Bio/NanoEntek. Both instruments feature a unique pipette tip electrode chamber instead of a standard electroporation cuvette. The performance of these instruments is comparable, but the Neon Transfection System has the following improved features:
- Improved user touchscreen interface with larger display area
- Most updated firmware (available at Instrument Management: https://www.thermofisher.com/us/en/home/products-and-services/services/instrument-qualification-services/instruments-and-services-portal.html)
- Pre-programmed with one 24-well optimization protocol
- Improved sensor connector design
- >130 validated cell-specific online protocols
We do not carry an adapter to allow compatibility between former and current devices. The kits and components in both systems are exactly identical except that they are branded as Neon in the Neon Transfection System. The old manual can be used for the Neon Transfection System instrument. More information regarding the history of the Neon Transfection System can be found here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/history-of-the-neon-transfection-system.html).
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The Neon Transfection System has the following unique advantages:
1. Pipette tip chamber design for easy cell handling, uniform electric field, and higher viability due to minimal pH change during electroporation.
2. Single transfection kit (Neon Kit) compatible with all mammalian cell types, including primary and stem cells, thereby avoiding the need to determine an optimal buffer for each cell type. Only two cell resuspension buffers cover all cell types: T buffer for primary T and B cells, PBMCs, monocytes, and bone marrow–derived cells, and R buffer for established adherent and suspension cells as well as primary adherent cells.
3. Easily scalable for small or large cell culture formats. Use as few as 1 X 10E4 or as many as 5 X 10E6 cells per electroporation in a sample volume of either 10 µL or 100 µL.
4. Over 130 validated online protocols, optimized for ease of use and simplicity. Visit our Neon Cell Database (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html)
5. Pre-programmed with a 24-well optimization protocol to quickly identify best electroporation parameters by cell type.
6. Flexible payload and applications. Delivers DNA, mRNA, siRNA, and proteins.
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Yes. We recommend verifying the integrity of your DNA on an agarose gel to see if it is degraded. Supercoiled plasmid runs faster than linear plasmid. Nicked plasmid will run slower than linear plasmid. The content of “nicked” DNA in your DNA preparation should be below 20%. Higher content of nicked DNA results in a significant decrease in transfection efficiency.
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Yes. To check the quality of your DNA, we strongly recommend confirming both A260:A280 and A260/230 ratios are between 1.6-2.0 and check for DNA degradation by agarose gel electrophoresis.
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No, the precipitation is irreversible. Please contact Technical Support at techsupport@thermofisher.com to obtain a replacement, if the Neon kit was purchased within 1 year.
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We recommend the following for large plasmid transfection using the Neon Transfection System:
- Prepare highly concentrated plasmid (i.e., 5 mg/mL) to keep DNA volume less than 10% of the total electroporation reaction.
- Use pure plasmid to avoid cytoxicity issues. Confirm that A260/280 and A260/230 ratios are between 1.6-2.0. For endotoxin-free plasmid DNA, we recommend the PureLink Expi Plasmid Purification kit.
- Keep DNA quantity to ranges recommended in the product manual for the 10 or 100 µL tip. Some optimization is normal based on variability in cell size, cell density, and plasmid.
- Confirm DNA integrity and absence of degradation by agarose gel electrophoresis.
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To determine the Neon transfection efficiency for siRNA, we recommend transfecting the cells with a fluorescent-labeled negative control siRNA (BLOCK-iT Fluorescent Oligo, Cat. No. 13750062) and measuring the transfection efficiency as the percentage of fluorescent stained cells among viable cells. However, keep in mind that there is a caveat with this approach: the transfection efficiency determined by fluorescent-labeled negative control siRNA may over-estimate the transfection efficiency, as fluorescence detection with a microscope does not distinguish the siRNA that enters the cell from the siRNA that may adhere to the cell membrane. Instead, we recommend transfecting with a positive control siRNA that targets a housekeeping gene such as GAPDH and measure mRNA or protein knockdown.
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There are several possible reasons for this. Monocytes and macrophages respond to very low levels of endotoxin (LPS), which could have been introduced with your plasmid DNA. Use plasmid DNA that has been purified by anion-exchange chromatography, such as our PureLink HiPure Plasmid. PureLink Expi, or Purelink Expi Endotoxin-Free Purification Kits. If you still observe activation, you may subject your plasmid to a second round of anion-exchange chromatography purification. If you still get activation, the plasmid itself may contain sequences that stimulate the production of Interferon gamma. It is also possible that certain components in your culture medium, including the FBS batch you are using, may cause activation. Please make sure that none of these components activates your cells. The procedure for isolating your monocytes is also important. We recommend negative rather than positive selection, as it leaves the monocytes “untouched” by antibodies. Our electroporation buffers are guaranteed to be endotoxin-free and do not cause monocyte/macrophage activation in our hands.
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Arcing could be caused by high voltage or pulse length settings, high salt or contaminants in the DNA sample, incorrect cell density, and bubbles formed during mixing of cell samples. We recommend performing the 24-well optimization (see product manual) to identify the best electroporation parameters for your cell type. Please ensure that plasmid DNA A260/280 and A260/230 ratios are between 1.6-2.0. Use either a hemacytometer or Countess II Automated Cell Counter to accurately determine cell density. Mix samples gently to avoid bubble formation and pipette samples in a slow, smooth motion to avoid air uptake.
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For initial measurement of siRNA uptake, our FITC-labeled BLOCK-iT Fluorescent Oligo for electroporation (catalog number: 13750062) can be used. However, the fluorescent signal is somewhat weak and it is not easy to determine whether the oligo has actually entered the cells or is just sticking to the outside of the plasma membrane. In addition the signal fades quickly. Therefore, a better strategy is to use qPCR to measure knockdown of an actual target mRNA such as GAPDH and normalize it to a scrambled negative control.
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They could get activated, and there are several possible reasons for this. Monocytes and macrophages respond to very low levels of endotoxin (LPS), which could have been introduced with your plasmid DNA. Make sure you use plasmid DNA which has been purified by anion exchange chromatography, such as our PureLink HiPure kits. If you did this and still see activation, perform one or two rounds of PEG precipitation to remove residual endotoxin.
Alternatively, you can subject your plasmid to a second round of anion exchange chromatography purification. If you still get activation, the plasmid itself may contain sequences which stimulate the production of Interferon gamma. It is also possible that certain components in your culture medium, including the FBS batch you are using, may cause activation. Please make sure that none of these components activates your cells.
The procedure for isolating your monocytes is also important. We recommend negative rather than positive selection, as it leaves the monocytes "untouched" by antibodies.
Our electroporation buffers are guaranteed endotoxin-free and do not cause monocyte/macrophage activation in our hands.
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DNA amount and quality are very critical for electroporation of Dendritic Cells (recommended 0.5-1µg; maximal 2 µg). LPS (lipopolysaccharides) have a strong negative effect on cell viability. Please make sure that all components you use for dendritic cell culture and transfection, e.g. PBS, FCS and especially DNA, are LPS free. We additionally recommend purifying your DNA by precipitating it twice with 20% PEG/2.5 M NaCl. Viability is usually not an issue when working with mRNA or siRNA.
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Our online protocol suggests to use 50,000 cells with the 10 microliter tip. However, HL-60 cells do not survive well when transfected at low density. To improve viability you can try using at least 100,000 (better: 200,000) cells with the 10 microliter tips. Also, make sure your plasmid DNA is highly purified, as HL-60 cells are sensitive to LPS. If you observe that viability is good after 24 hours but decreases over the next 72 hours, you may either be using the wrong culture medium (RPMI 1640 + 10% FBS is recommended, do not use DMEM) or your batch of FBS contains low levels of cytotoxic contaminants.
Other possibilities are mycoplasma contamination or very high passage number of your cells. If this is the case please buy a fresh vial of HL-60 cells from ATCC and try your transfections again.
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There is no reason to speculate that an optimized electroporation parameter would be different between 10 ul and 100 ul tips. Therefore, even though the table in the Neon cell database states 10 ul tip, those conditions should be OK for 100 ul tip also as long as the density of the cells remains the recommended one. And if the result of 100 ul is much worse than 10 ul, change voltage either increasing or decreasing by 50 to 100V. It should work well with this slight adjustment of voltage.
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For large plasmids, it is important to prepare a highly concentrated solution of the plasmid for electroporation. An example: For the control plasmid, 0.5 ug is used for a 10 ul electroporation. The size of the control plasmid is about 5.5 kb. If your large plasmid is 50 kb, then the size is almost 10 times larger than the control and you have to use 10 times more weight of plasmid to have an equal number of molecules present. So you will need 5 ug of the large plasmid in a 10 ul electroporation to have an equivalent number of plasmids present. In order to keep the volumes low, the plasmid concentration will have to be over 5 mg/ml.
One thing to keep in mind is that when you add high amount of plasmid, it can damage cells due to toxicity of plasmid sample itself. So you will need to optimize the plasmid amount. A recommended strategy would be to start by adding the 50 kb example plasmid up to 2 ug per each 10 ul electroporation. Even though theoretically you should add more than 2 ug due to the large size of plasmid, start with 2 ug to avoid any toxicity. Then check the result. If viability looks OK and efficiency is lower than expected, increase the amount of plasmid in further reactions until you find the optimal amount for best results.
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No, the precipitate that forms in this buffer is irreversible.
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Yes. To check the quality of your DNA, we strongly recommend determining the A260:A280 ratio. It should be at least 1.6 for a good DNA preparation.
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Yes. You should verify the integrity of your DNA on an agarose gel to see if it is degraded. Supercoiled plasmid runs faster than linear plasmid. Nicked plasmid will run slower than linear plasmid.
The content of "nicked" DNA in your DNA preparation should be below 20%. Higher content of nicked DNA results in significant decrease of transfection efficiency.
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Here are some possibilities:
(1) Sub-optimal electrical parameters
(2) Poor plasmid quality such as endotoxin contamination
(3) Plasmid preparation containing high salt
(4) Plasmid quantity too high
(5) Cells are stressed or damaged
(6) Multiple uses of the same Neon tip
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Here are some possibilities:
(1) Sub-optimal electrical parameters
(2) Plasmid preparation containing high salt
(3) Plasmid larger than 10 kb
(4) Plasmid concentration too low
(5) Cells are stressed, damaged or contaminated by Mycoplasma
(6) Cell density too low or too high
(7) Cells with high passage number
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You cannot. You need to lift the cells off of the plate through a normal method such as that used to “passage” the cells (e.g., trypsin), perform the transfection in the Neon tip, and re-plate the cells in the plate after transfection.
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The optimal time for protein analysis is related to the stability of the protein being expressed. For a short-lived protein, like luciferase, analysis should be done at 6-18 hours after electroporation. For a more stable protein, such as GFP, you should start the analysis at 24 hours or longer post-electroporation.
As electroporation uses electric shock to make pores on cell membrane, this can damage certain membrane proteins. But those membrane proteins will generally be re-expressed and recover as time goes by. The recovery time may vary and will depend on the type of cell and protein; there is no general guideline for this yet.
An exception are primary cells which do not proliferate after electroporation, where the membrane damage could be permanent such that it hinders certain membrane protein studies. However, this is quite rare. Given sufficient time to recover, for most proliferating cells there should be no problem with membrane proteins.
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No. This technology was originally developed by a Korean company named Digital Bio Technology and marketed under the name “microporation”. Since its inception in 2006 there have been over 40 publications describing the use of this electroporation technology. Please go to www.microporation.com for further information. Invitrogen is now the only provider selling a re-designed microporator under the name Neon Transfection System.
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After electroporation, wait for about 4-6 hours before adding antibiotics back to the cells. This is to make sure that the membrane integrity has been restored.
You need T buffer instead of the standard R buffer for primary T and B cells. These cells are smaller than regular cell types and require higher voltage for successful electroporation. If you use R buffer and apply high voltage, you will see sparks. With R buffer, voltage over 1800V generally generates sparks regardless of cell number and other condition. The maximum voltage for R buffer is around 1900V. In order to apply higher voltage, you need a buffer less conductive than R buffer, which is the T buffer.
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Buffer T is an alternative cell resuspension buffer for use with primary blood cells. Its composition differs from that of Buffer R and allows the application of higher voltages due to lower conductivity. Buffer T works with T-cells, B-cells, monocytes, and PBMCs as well as bone marrow-derived cells. It does not work with established cell lines or primary cells which have been kept in culture for some time. In some situations it is not immediately clear whether Buffer R or Buffer T would work, so both should be tried in separate optimization experiments.
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In most cases instrument settings for a certain cell line or primary cell type which were optimized using a plasmid will also work with siRNA. However, those settings may not be the best possible settings for the delivery of siRNA. Therefore, additional optimization may be required to improve knockdown efficiency of the target. For cell lines or primary cell types which have not been optimized with plasmid DNA, a 24-well optimization is the best approach to find efficient conditions. Keep in mind that for every condition tested, a negative control siRNA needs to be transfected in order to normalize knockdown efficiency.
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In most cases they can, but in some cases reduced transfection efficiency may be observed and adjustment of the voltage settings may be required to improve efficiency. A 24-well optimization with the 100 microliter tips is usually not necessary.
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According to Neon guidelines, you can run up to 8 million cells in 100 µL. If the cells are small enough, up to 10 million cells can be used in 100 µL.
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We have transfected as few as 30,000 cells using the 10 microliter tips. If the cell density is too low during electroporation, viability will typically be compromised. This effect is somewhat cell type-dependent. Therefore, how low you can go with your cell line or primary cell type without substantially reducing viability needs to be determined empirically. One of our customers has reported successful transfection of 5000 primary hair follicle cells.
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We have data which shows knockdown of EmGFP by a specific siRNA which was co-transfected with an EmGFP expressing plasmid, as well as knockdown of endogenous genes.
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We currently do not have data to support this application, but co-transfection of different plasmids is possible. However, the amount of DNA should be carefully titrated since overloading the cells with plasmid DNA or using unfavorable ratios of the plasmids may cause toxicity. Therefore, we recommend starting optimizations of co-transfection experiments with low amounts of total DNA followed by a stepwise increase. Various ratios of the plasmids should be tested if toxicity is observed
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The electrolytic buffer tubes are disposables and you should change the tubes when changing cells in order to avoid cross contamination. With regard to plasmid DNA, the answer depends on the sensitivity of your downstream assay. If slight plasmid cross-contamination does not affect your assay, you may not have to change the tubes. Please perform pilot experiments to determine whether it works for you. However, we still recommend changing the electrolytic buffer. In case you run out, the tubes can be purchased separately (Catalog number: MPT100).
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Go to our "Learn More about the Neon Transfection System" page at www.thermofisher.com/neon. There you will find a link to our cell transfection database which currently contains in-house optimized settings for over 140 cell lines and primary cells. However, these conditions may have to be modified slightly for your particular cell line since passage number and/or culture conditions or cell isolation procedures may not be the same as ours. The conditions which we have posted on our website should be understood as a starting point for your own optimization.
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To avoid contamination caused by carry-over from one sample to another, we recommend that you do not use the Neon Tip for more than 2 times. Oxide formation at the piston surface area can be generated if the tips are used more than 2 times, which decreases electrode function of the piston.
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We recommend using the tips twice. The reason for this recommendation is that the wire electrode inside the tip is coated with 24 karat gold. Some of the gold is released each time an electrical pulse is delivered. Therefore, repeated use of the tip will result in the gold coating becoming thin and causing conductivity of the tip to change. If you want to be absolutely sure that the correct voltage and current are delivered to your cells, use the tip only twice.
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We typically stain suspension cells with 0.5% propidium iodide, then gate viable and non-viable populations using a Guava 96-well PCA (Millipore). Transfection efficiency obtained with our EmGFP-expressing control plasmid is calculated based on the total population of cells. This is in contrast to Amaxa which calculates efficiency with respect to the viable cell population. For adherent cells, we trypsinize and then pool trypsinized and non-adherent cells from the supernatant prior to staining with propidium iodide.
If you do not have a Guava PCA system or FACS, you can determine viability and transfection efficiency using the trypan blue exclusion method and a fluorescence microscope with filter settings appropriate for the detection of GFP (emission: 509 nm). However, this approach is less accurate than FACS and much more tedious.
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There is no evidence that larger plasmids cause increased toxicity in all cases. The toxic effects that are seen with some large plasmids are not related to their size, but are likely due to sequences located on the plasmid or to contaminants in the plasmid preparation, such as LPS. Always use anion exchange chromatography-based kits (such as Invitrogen’s PureLink HiPure kits) to prepare transfection grade plasmid DNA and avoid overloading the columns, as this will result in plasmid preparations of low purity.
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We routinely use plasmids of 4-7 kb in our laboratories and plasmids up to approximately 20kb should not be a problem. Using plasmids larger than this will most likely result in lower transfection efficiency. Some preliminary results we have indicate that very large BAC's can be transfected as well, but also with a low transfection efficiency.
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As the stability and half-life of various mRNAs and their protein products varies, it is important to empirically determine the best time points for assessing target knockdown. For example, it has been documented that in mammalian cells, mRNA half-life can range from minutes to days (Ross J, 1995, Microbiol Rev 59:423-50) while the half-life of protein products can range from less than a few minutes to several days. In general, the recommended time course ranges from 12 to 72 hours to deplete target mRNA and 24 to 96 hours to adequately knock down target proteins.
For each cell type in our cell database, we offer thoroughly optimized electrical parameters along with a universal electrolytic buffer. Very little optimization is required. For cells that are not listed in our database, there is a pre-programmed optimization protocol built in the Neon device.
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A good start is to use the plasmid electroporation parameters for the same cell type. The Neon Cell Database (www.thermofisher.com/neon) contains optimized plasmid electroporation parameters for many commonly used cell types (the list is growing). If the Neon Cell Database does not contain the cell type of interest, you can use the 24-well optimization protocol that is preloaded on the Neon device.
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To determine the Neon System transfection efficiency for siRNA, the cells are transfected with a fluorescent-labeled negative control siRNA; the transfection efficiency is measured by the percentage of fluorescent stained cells among all cells, both live and dead. We recommend to use the BLOCK-iT Fluorescent Oligo for electroporation from Invitrogen (Cat. No. . 13750062). However, there is a caveat with this approach: the transfection efficiency determined by fluorescent-labeled negative control siRNA may over-estimate the transfection efficiency, as fluorescent detection with a microscope does not distinguish the siRNA that goes into the cell from the siRNA that sticks to the cell membrane. To obtain a more accurate transfection efficiency, one needs to transfect the cells with a positive control siRNA such as the one that targets a house-keeping gene and measure the knockdown of target RNA or protein.
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The main concern for multiple use of the Neon Tube is the possibility of cross-contamination. In addition, we strongly recommend that a new Neon Tube be used for each different plasmid DNA/siRNA or cell type.
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(1) If you find another cell type in the Neon Cell Database that is similar to your cell type in terms of tissue origin, you can use the parameters of that cell type for your cell type. You may not get optimal results, but it is a good starting point. One example: your have 293 T cells and you find HEK293 cells in the Neon database. Since both are derived from human embryonic kidney, you can use the electroporation parameters of HEK293 cells for 293 T cells.
(2) You can use the preprogrammed 24-well optimization protocol in the Neon device to optimize conditions for your cell type
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If the cell type has worked with electroporation, it should work with the Neon System.
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Circular and linearized plasmids which do not contain special recombination sequences generally transfect with the same efficiency and integrate into the genome with similar probability. However, the area of recombination on the plasmid can be influenced by linearization, as loose ends are preferred over continues stretches of sequence. By linearizing the plasmid, you can thus determine at which position within the plasmid the recombination occurs, thereby conserving the expression cassette in most cases.
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While we would expect this to be possible, we do not have in-house data to support this application.
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We recommend using anion exchange chromatography to prepare transfection grade plasmid DNA. This technology is found in our PureLink HiPure plasmid purification kits. Do not use standard mini-prep spin columns, as they contain silica membranes which do not remove impurities to the same extent as anion exchange resins.
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Unlike standard cuvette-based electroporation chambers, the Neon system uses a biologically compatible pipette tip chamber. The design of a gold coated wire electrode inside a pipette tip has been shown to produce a more uniform electrical field and a lower pH gradient across the cell suspension. Therefore, this design allows for a better maintenance of physiological conditions resulting in very high cell survival compared to conventional electroporation*.
* Kim JA, Cho K, Shin MS, et al. (2008) A novel electroporation method using a capillary and wire-type electrode. Biosens Bioelectron 23(9):1353-1360.
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Yes. The Neon Transfection System can be used for any RNAi substrate (siRNA, shRNA, miRNA). You can use the same conditions described in the cell type-specific protocol for DNA, or use the pre-programmed 24 step optimization protocol.
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Cell fusion may occur "accidentally" as a side-effect during transfection for some cell-types which tend to form cell clusters (e.g. PC-12 cells), but unfortunately, we do not offer a Neon system program to optimize for cell fusion.
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The Neon Transfection System efficiently delivers nucleic acids (DNA/siRNA) into most mammalian cell types, including traditionally difficult-to-transfect cells like primary cells and neurons. In comparison, lipid transfection reagents are much less efficient for such cell lines, and in some cases do not work at all. While viral delivery is also generally effective for difficult-to-transfect cells compared to lipids, it can be much more labor intensive and time consuming than electroporation with the Neon Transfection System, especially if you have many different DNA or RNAi molecules to transfect and screen.
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We normally analyze transfection efficiency in living cells by FACS. We first exclude cellular debris by gating for the "normal" population (regarding size and granularity) in the forward-sidescatter. From this gated population we determine dying cells by propidium iodide staining and exclude them from analysis by setting another gate. For example, in one experiment we transfected Human primary CD4+T-cells with 2250V, 20ms, 1 and 0.5 ug EGFP in primary blood buffer using the Neon System. 24 hrs post electroporation, cells were analyzed by flow cytometry. CD4+T-cell were gated according to forward/side scatter, and dead cells were excluded by propidium iodide staining and gating. GFP gene expression of T cells was measured after electroporation with plasmid DNA, and we found 32.74% transfection efficiency and 75% viability. Please see our "Learn More about Neon Transfection System" pages (search this term from our home page) for more data and information.
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Cells were analyzed for viability and transfection efficiencies 24 hours or 48 hours post-electroporation using a Guava PCA-96 Cell Analysis System. LIVE/DEAD cell viability assay populations were calculated by propidium iodide staining (1:2000). The percent of transfected cells was calculated by dividing the number of GFP positive cells by the total population and recorded as transfection efficiencies. The percent of dead cells was calculated by dividing the number of PI-stained cells by the total population
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The Neon system measures the transfection efficiency by the percentage of GFP-positive cells among all cells which include live and dead cells. In contrast, the Nucleofector Device measures the transfection efficiency by the percentage of GFP-positive cells among only the live cells.
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Clean the surface of the Neon device and Neon Pipette Station with a damp cloth. Do not use harsh detergents or organic solvents to clean the unit. Avoid spilling any liquid inside of the Neon Pipette Station. If you accidentally spill any liquid (e.g., buffer, water, coffee) inside the Neon Pipette Station, disconnect the station from the main device and wipe the station using dry laboratory paper. Invert and leave the station for 24 hours at room temperature for complete drying. Do not use an oven to dry the Neon Pipette Station
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(1) Small to large number of cells can be used. The transfection is performed using as few as 2 x 10E4 or as many as 8 x 10E6 cells per reaction using a sample volume of 10 ul or 100 ul.
(2) The Neon Transfection System uses a single transfection kit (Neon Kit) that is compatible with various mammalian cell types, including primary and stem cells, thereby avoiding the need to determine an optimal buffer for each cell type. Two cell suspension buffers cover all cell types: T buffer (not yet included in the kit) for primary T and B cells, and R buffer for other cells.
(3) Open and transparent protocols that are optimized for ease of use and simplicity. The Neon Cell Database (www.thermofisher.com/neon) contains optimized protocols for many commonly-used cell types.
(4) The Neon device is preprogrammed with one 24-well optimization protocol to optimize conditions for your nucleic acid/siRNA and cell type.
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We do not offer such a service at this time. The Neon Transfection System is designed to facilitate the optimization of transfection conditions. Typically, three rounds of optimization are sufficient to find the best instrument settings for any given cell line or primary cell type. Unless you prepare your cells from very small amounts of tissue, or tissue which is difficult to process, optimizing your cells should not take more than a week and costs a lot less than a custom service.
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Our control plasmid is a 5.9 kb pcDNA6.2-based expression construct expressing EmGFP from a CMV promoter. The purification procedure is proprietary, but the purity of this plasmid is equivalent to two rounds of anion exchange chromatography.
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We do strongly advise against washing the tips. Washing will not remove DNA or siRNA attached to the tip and will increase the risk of cross-contaminating your samples. The tips can not be sterilized easily after cleaning, thus increasing the risk of microbial contamination of your cultures.
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Yes, they do.
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The Neon System is simply a next generation Electroporation Technology system. The main difference between the two systems is the user interface. The kits and components are exactly identical except they are branded as Neon Kits
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All buffer compositions are proprietary.
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The E2 buffer has higher osmolarity than E buffer. Higher osmolarity prevents the leakage of electroporation content from the 100 ul Neon tip which has a larger hole at the tip end than the 10 ul Neon tip (pore diameter of the Neon tips: 100 ul tip = 2.10 mm; 10 ul tip = 0.65 mm).
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The “R” buffer is used to resuspend the cells prior to transfection (“R” for Resuspension”). The “E or E2” buffers are the electrolytic buffers used for electroporation. “E or E2” buffers are added to the Neon tube prior to electroporation
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There is no expiration date for the Neon Transfection System 100 μL Kit (Cat. No. MPK10096, MPK10025). However, Thermo Fisher Scientific does warranty the product for 12 months after it has been shipped to the customer.
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No, the tips within the Neon Transfection System 100 μL Kit (Cat. No. MPK10096, MPK10025) are not available as a stand-alone product.
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We recommend the following guidelines:
- For high transformation efficiencies, the OD750 of the C. reinhardtii culture should be > 0.8 before electroporation.
- Carry out all transformation steps at 4 degrees C using solutions pre-equilibrated at 4 degrees C.
- Electroporate the cells using the 100 µL Neon tip in TAP-40 mM sucrose solution or the MAX Efficiency Transformation Reagent For Algae (Cat. no. A24229) at 4 degrees C.
- Use the following Neon electroporation parameters: 2300 volts (Voltage), 13 ms (Pulse Width), 3 (Pulse Number)
For detailed instructions on using the Neon Transfection System, refer to the Neon Transfection System User Guide (https://tools.thermofisher.com/content/sfs/manuals/neon_device_man.pdf).
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