Search
Search
View additional product information for CytoTune™-iPS 2.0 Sendai Reprogramming Kit - FAQs (A16518, A16517)
94 product FAQs found
丙戊酸通常被认为能够提升慢病毒等整合性病毒系统的重编程效果。但其对CytoTune试剂所介导的重编程似乎没有促进效果，因为仙台病毒是一种非整合性的RNA病毒。
CytoTune-iPS 2.0仙台病毒重编程试剂盒包含了温度敏感的c-Myc和KOS变异的载体，能够更方便地实现清除操作。如需清除c-Myc和 KOS，请将iPSC细胞在38–39°C孵育5天。用户需要注意iPSC细胞的敏感性，因此我们只推荐对传代10次以上仍含有仙台病毒，且已通过RT-PCR技术确认过您的细胞中不含 Klf4 载体（这个载体不含温度敏感性的突变）的iPSC细胞系执行升温操作，这样您就可通过升温来去除c-Myc和 KOS基因了。
基于您所用细胞类型，可预期在转导后24-48小时观察到某种程度的细胞毒性，50%以上的细胞都可能受到影响。这表明细胞接受了大量病毒，并表达外源基因所致。我们推荐您按照实验方案中的步骤进行持续培养。
TaqMan仙台病毒基因检测Assay和CytoTune 2.0和CytoTune 2.1版本都兼容，但有一个例外。CytoTune 2.0试剂盒包含一个表达c-Myc的载体，而CytoTune 2.1试剂盒包含的是表达L-Myc的载体。因此，对每种试剂盒应使用与之相对应的Assay(例如，SEVCMYC用于CytoTune 2.0, SEVLMYC用于CytoTune 2.1)。所有其它Assay对这两种试剂盒都是兼容的。
我们没有用于终点法RT-PCR的这种引物。然而，我们确实为每一个外源性重编程基因提供了经过验证的TaqMan基因表达Assay (https://www.thermofisher.com/order/genome-database/browse/gene-expression/keyword/sendai+taqman)。这些Assay被设计用来专门检测仙台病毒载体上发现的外源基因的表达，不会与这些基因的内源性表达发生交叉反应。此外，这些Assay也可被用于检测每种CytoTune仙台病毒载体的存在，以确定这些载体是否从已建立的ipsc中清除了。
对于包含3种载体（KOS，cMyc，Klf4）的Invitrogen CytoTune 2.0试剂盒而言，cMyc与KOS载体均能通过升温至39°C去除。升温对于cMyc载体更为有效，对KOS载体也能起效。
注意：在开始升温之前，请首先验证单独的Klf4载体已被清除。Klf4载体不会被升温操作所去除，如果在Klf4仍存在的条件下进行升温会导致某些问题。
您可通过多种方法确认细胞中仙台病毒的存在，包括使用抗仙台病毒的抗体进行染色，或使用CytoTune-iPS 2.0仙台病毒重编程试剂盒用户手册 (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf)第50页上所列的引物对进行终点法RT-PCR检测，或使用TaqMan Sendai Gene Expression assays（也列在说明书第50页上）进行实时RT-PCR检测。
病毒从细胞中清除可以少至5代或多至15代。在极少数情况下，仙台病毒序列可以持续存在。清除率与克隆相关，可通过PCR或抗仙台病毒抗体确认。有关生成无载体的iPSC的更多信息，请参阅用户手册（https://tools.thermofisher.com/content/sfs/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf）。
为了维持iPSC的健康状态，须对这些细胞进行监控并每天更换生长培养基。通常情况下，在细胞达到70-80%汇合度或多数克隆直径大于700 μm时，应对iPSC克隆进行传代。请参考用户手册（https://tools.thermofisher.com/content/sfs/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf）获取完整的实验方案。
我们在使用该一抗与二抗组合的过程中没有发现任何问题。IgG二抗会与IgM一抗发生交叉反应，因为IgM的Kappa轻链与IgG的相同。
手册中列举的IgG二抗均可供使用，它们能够有效识别IgG一抗和IgM一抗等抗体。基于以上理由，我们一般常备这些IgG二抗，并在绝大多数应用中使用它们。这就是推荐它们的主要原因。
请注意Invitrogen CytoTune-iPS仙台病毒重编程试剂盒（货号A13780-01，A13780-02）已经停产。
iPSC克隆可通过碱性磷酸酶染色法轻松实现可视化，相关试剂盒有碱性磷酸酶活细胞染色试剂盒（Alkaline Phosphatase Live Stain，货号 A14353）。此外，重编程克隆可通过Tra1-60或Tra1-81抗体的活细胞染色来进行筛选，这些抗体能够识别未分化的iPSC，进而帮助用户从各种人体细胞中鉴定出重编程细胞。请参考用户手册（https://tools.thermofisher.com/content/sfs/manuals/AP_Live_Stain_man.pdf）获取完整的实验方案。
iPSC的预期生长形态应具有边界清晰，紧密聚集的细胞克隆和高核质比。如您未观察到这些形态，或所观察到克隆数目较少，则应提高转导时的MOI值。
我们不推荐在使用Invitrogen CytoTune-iPS 2.0仙台病毒重编程试剂盒的过程中使用VPA。
Invitrogen CytoTune-iPS 2.0仙台病毒重编程试剂盒能够为BJ成纤维细胞提供0.02-1.2%的重编程效率。这一参数在其他种类的细胞上可能会有所改变。
您可按照冻存任意多能干细胞的实验方案来冻存iPSC。推荐使用含10% DMSO的生长培养基来冻存细胞。或使用我们的PSC冻存试剂盒（货号 A2644601）。
iPSC克隆在转导后3周左右开始逐渐形成。只需载体的一次性应用即可介导重编程成功，转导后21-28天就可以开始筛选iPSC克隆了。
请避免对重编程载体进行反复冻融。每一次冻融循环都可能会造成病毒滴度的显著下降，经过反复冻融的试剂盒的使用效果无法保证。
重编程的细胞可生长于标准的iPSC培养基中。我们推荐在有饲养层的条件下使用添加Gibco KnockOut血清替代物（KSR）的培养基，或在无饲养层的条件下使用Gibco Essential 8培养基。请参考用户手册获取完整的实验方案。
如果您希望使用Invitrogen CytoTune 2.0对PBMC进行无饲养层条件下的重编程，您可按照现有的Invitrogen CytoTune 2.0 PBMC方案来操作，不过需要在第三天将细胞种植于Gibco玻连蛋白或Geltrex基质中代替MEF，之后在第7-8天以Gibco Essential 8代替Gibco KnockOut基于血清替代物的PSC培养基。
这时的重编程效率通常会比基于饲养层条件下的效率低一些，不过您仍可获得一些阳性克隆。
我们推荐首先尝试只增加hKlf4的MOI值。举例：从5:5:3的比例调整为5:5:6。如果仍需优化，再增加KOS与hc-Myc的MOI值。KOS与hc-Myc之间的比例应保持1:1，hKlf4的MOI可独立改变。例如：从5:5:3的比例调整为10:10:3或10:10:6。
推荐对大多数细胞类型使用MOI=5:5:3（KOS，hc-Myc，hKlf4）的条件。不同批次产品的病毒滴度有所不同；实现每一MOI值所需的体积均列于每批产品的分析认证书（CoA）中。由于MOI值随细胞类型而变，您也可基于细胞类型来优化您的MOI值。KOS与hc-Myc之间的比例应保持1:1，hKlf4的MOI可独立改变。举例：如果KOS的MOI为4，则hc-Myc也一定为4。
MOI（感染复数）是描述病毒粒子与细胞之间比值的一个参数。Invitrogen CytoTune 2.0试剂盒三种载体中的每一种都应基于推荐的MOI值来加入细胞中。我们推荐用户按照以下MOI值作为起点，若重编程效率不佳可进行调整：
Invitrogen CytoTune 2.0 KOS：起始MOI为5
Invitrogen CytoTune 2.0 hc-Myc：起始MOI为5
Invitrogen CytoTune 2.0 hKlf4：起始MOI为3
对于每个孔均按照2 x 10E5至3 x 10E5个细胞/孔的推荐密度种植人体真皮成纤维细胞的6孔板而言，一个试剂盒（每种载体一管，即总计3管）足够以MOI=5:5:3（KOS，hc-Myc，hKlf4）的比例转染其中的5个孔。病毒仅能使用一次，因为每个冻融循环都会造成病毒滴度的显著降低。
我们推荐您仅使用该病毒一次，因为每个冻融循环都会造成病毒滴度的显著下降。
是的，您必须同时使用全部三种重编程载体。省略其中的一或两种载体很可能造成重编程效率极低乃至失败。
这些试剂盒一经收货，就应保存在–80°C条件下。
该病毒会传给子代细胞，其浓度也会随着时间而逐渐降低。病毒不会离开细胞和再感染其它新细胞，因为融合基因已经从病毒基因组中删除。
尽管人类并非SeV的天然宿主，该病毒对人体也无致病性，不过必须采取适当的保护，以避免发生与该病毒潜在的粘膜接触。Invitrogen CytoTune-iPS 2.0仙台病毒重编程试剂盒必须在包含生物安全柜和层流罩的二级生物安全（BL-2）防范条件下使用，并使用适当的个人安全设施以避免粘膜暴露/泼溅情况的发生。
是的。我们已成功地使用CytoTune-iPS 2.0 仙台病毒重编程试剂盒从小鼠成纤维细胞诱导产生出iPSC。
是的。请在此处查询 实验方案（https://www.thermofisher.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols.html）或参考文献（https://www.thermofisher.com/us/en/home/life-science/stem-cell-research/stem-cell-research-resources/stem-cell-research-publications.html）。
我们的重编程试剂盒经验证对于许多种类的细胞有效，其中包括人成纤维细胞，CD34阳性的脐带血细胞，和外周血单核细胞（PBMC）。如需查阅当前已验证本方法有效的细胞类型的引用文献列表，请点击此处（https://www.thermofisher.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/sendai-virus-reprogramming/cytotune-publications.html）。
非整合型的重编程方法能够生成不含可检测到载体或外源基因的iPSC。传统的重编程载体（即慢病毒，逆转录病毒）会整合进入靶标细胞的基因组之中。随之产生的iPSC及这些iPSC分化出来的细胞都会包含外源DNA，因此用于细胞治疗和药物发现等方面都可能造成麻烦或存在安全性方面的问题。此外，整合过程还可能发生在基因组的关键区域，进而导致无关的发育进程出现问题。
Invitrogen CytoTune-iPS 2.0仙台病毒重编程试剂盒中的仙台病毒载体是基于一种经修饰，不具有传染能力的SeV进行设计的，其中的融合基因（F）已经被删除。病毒载体仍保留了对广泛细胞的全部感染能力；不过由于病毒基因组中缺乏F基因，它们已无法在被侵染细胞中产生感染性的病毒粒子。仙台病毒载体转入了能够翻译出hOct3/4，hSox2，hKlf4和hc-Myc蛋白的基因。病毒载体在转导后会使细胞表达这四个基因，从而介导重编程过程。
仙台病毒，也称为日本血凝病毒（HVJ），是在1950年代初期在日本仙台首次分离到的小鼠和大鼠呼吸道病毒。该病毒属于副粘病毒科的I型小鼠副流感病毒。SeV是具有包膜的病毒，直径为150-250 nm，其基因组是单链的反义RNA（含15384个碱基）。这种病毒通过与各类细胞表面呈递的唾液酸受体相结合进而感染细胞，因此能够广泛感染各类动物的多种细胞。
可以，用户可通过额外添加Klf4载体对系统进行微调。增加Klf4的用量能够提升重编程效率，而减少其用量能够有助于降低病毒的总量。
KOS的多顺反子设定中需要额外的聚合酶活性补偿同一载体上的三种基因组合。Invitrogen CytoTune-iPS 2.0系统以hKlf4载体提供额外的聚合酶活力，驱动所有载体的重编程进程并提升重编程效率。此外，增加hKlf4的表达也能够提升重编程效率。
KOS是基因组合hKlf4，hOct3/4，hSox2的首字母缩略词。这是一个多顺反子载体，即所有这三个基因的阅读框都包含于同一载体中。
Invitrogen CytoTune-iPS 2.0试剂盒（货号A16517，A16518）相比已停产的原始Invitrogen CytoTune-iPS仙台病毒重编程试剂盒（货号A13780-01，A13780-02），能够为用户提供更高的重编程效率，更快的载体清除率，以及更低的细胞毒性。
2.0试剂盒包含了三个载体，其中一个是专为增强重编程效率而设计的多顺反子载体（Invitrogen CytoTune 2.0 KOS）。这一多顺反子载体具有与众不同的骨架——在聚合酶相关基因中包含了温度敏感的突变，这一突变能够帮助用户在重编程操作完成后更快地清除病毒，对细胞毒性也更小。
Invitrogen CytoTune-iPS仙台病毒重编程试剂盒（货号A13780-01，A13780-02）是一套非整合型的系统，能够将体细胞重编程为诱导性多能干细胞（iPSC）。本试剂盒利用四种仙台病毒载体，分别表达Yamanaka四因子中的一种：Oct3/4，Sox2，Klf4与c-Myc。这些转录因子在体细胞中的表达被证明是成功诱导iPSC的关键因素。
请注意Invitrogen CytoTune-iPS仙台病毒重编程试剂盒（货号A13780-01，A13780-02）已经停产，并由Invitrogen CytoTune-iPS 2.0仙台病毒重编程试剂盒（货号A16517，A16518）。
是的，我们测试过并发现只有KOS（三合一），cMyc和Klf4的载体组合具有最高的重编程效率。举例来说，单独转染KOS和cMyc不足以诱导重编程。加入Oct4或Sox2只能获得非常少量的重编程克隆。这很可能是由重编程因子的化学剂量不平衡所致，这种不平衡可能会显著削弱重编程效率。
Invitrogen CytoTune-iPS 2.0仙台病毒重编程试剂盒（货号A16517，A16518）是一套非整合型的系统，通过仙台病毒载体将体细胞重编程为诱导性多能干细胞（iPSCs）。Invitrogen CytoTune-iPS 2.0仙台病毒重编程试剂盒包含三种Invitrogen CytoTune 2.0重编程载体，其中包括了Yamanaka四因子，Oct3/4，Sox2，Klf4和c-Myc。这些转录因子在体细胞中的表达被证明是成功诱导iPSCs的关键因素。成功的重编程操作只需这些载体的一次应用即可实现。
人iPS与人ES细胞的生长条件相同。与人ES细胞类似，人iPS细胞能够生长于辐照的MEF滋养层细胞上+含20% KnockOut SR，0.1 mM NEAA，1mM谷氨酰胺，0.1mM b-ME的DMEM/F12培养基 + 辐照MEF饲养细胞 + 100 ng/mL bFGF的培养条件中，或Matrigel基质包被培养皿 + MEF条件培养基 + 100 ng/mL bFGF的培养条件中（Science318:1917 (2007)）。人ES细胞与iPS细胞两者均可生长于KnockOut ESC/iPS培养基中（货号A14131）。
用于细胞培养：人重组激活化素A（货号PHC9564），bFGF（货号PHG0261），IGF-II（货号PHG0084）（Stem Cells 24:1476 (2006); Nature 448:1015 (2007)）。
用于人ES细胞分化：BMP-4（货号PHC9533），EGF（货号PHG0311）和HGF（货号PHG0324）（Proc Natl Acad Sci U S A 97:11307 (2000)）。
我们将本品的使用结果与Gibco Essential 8培养基搭配Invitrogen CytoTune-iPS 2.0 仙台病毒重编程试剂盒的实验结果进行了比较，未发现重编程效率方面受到明显影响。我们推荐在克隆形成阶段每天进行换液（第7-28天）。
The shelf life for the CytoTune-iPS 2.0 Sendai Reprogramming Kit is 2 years when stored at -80 degrees C with no repeated freeze-thaw cycles.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
We recommend reprogramming patient cells at the earliest passage possible. However, it is important to have the cells growing and healthy, which can take between 1-4 weeks. The cells are usually ready to reprogram once they have gone through a total of 3-4 passages.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Yes. Initial experiments with fibroblasts have shown that scaling down to a 12-well or 24-well works, but at a potentially reduced efficiency. Cell seeding densities may need to be optimized.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
We recommend seeding approximately 50,000-100,000 fibroblast cells per well of a 6-well to yield 200,000-300,000 cells two days later. This equals a confluency of 50-80% on the day of transduction. Overly confluent cells will result in a decreased transduction efficiency. When cells are overly confluent (>80% confluent), we recommend re-plating the cells to achieve a range of 50-80% confluency on the day of transduction.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The CTS CytoTune-iPS 2.1 Sendai vector formulation does not contain the bovine serum albumin carrier protein. Additionally, the preparation contains no animal-derived components at the primary component level. Each virus (i.e., KOS, L-Myc, and Kfl4) comes in a volume of 200 µL instead of 100 µL. The titer of the CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit can range from 0.5 x 10E8 to 2.0 x 10E8 virus particles per mL whereas the titer of the CytoTune-iPS 2.0 Sendai Reprogramming Kit is typically around 1.0 x 10E8 virus particles per mL.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Valproic acid is typically thought to enhance reprogramming with integrating viral systems such as lentivirus. It is unlikely to enhance CytoTune reagent reprogramming since Sendai virus is a non-integrating RNA virus.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The Invitrogen CytoTune-iPS Sendai 2.0 Reprogramming Kit contains a temperature sensitive mutant of c-Myc and KOS that facilitates the clearance of these vectors. To clear c-Myc and KOS, incubate the iPSCs at 38-39 degrees C for 5 days. One caveat is that given the sensitive nature of iPSCs, only perform this temperature shift if Sendai virus is in your iPSC lines after more than 10 passages, and you have performed RT-PCR to show that the Klf4 vector is absent from your cells (this vector does not have temperature-sensitive mutations). Then you can perform temperature shift to remove the c-Myc and KOS vectors.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Depending on your cell type, you should expect to see some cytotoxicity 24-48 hours post-transduction, which can affect >50% of your cells. This is an indication of high uptake of the virus and is caused by the expression of exogenous genes. We recommend that you continue culturing your cells and proceed with the protocol.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
AP is a phenotypic marker of pluripotent stem cells (PSCs), including undifferentiated embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryonic germ cells (EGCs) and Embryonic Carcinoma Cells (ECCs). While AP is expressed in most cell types, its expression is highly elevated in PSCs. Therefore, AP staining has been used to differentially stain PSCs to easily distinguish them from mouse embryonic fibroblasts (MEFs) used as feeders and parental fibroblasts commonly used in reprogramming experiments.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The TaqMan Sendai Gene Expression Assays are compatible with both CytoTune 2.0 and CytoTune 2.1, with one exception. The CytoTune 2.0 kit contains a vector which expresses c-Myc, while the CytoTune 2.1 kit contains a vector which expresses L-Myc. As such, the relevant assays should be used in conjunction with each kit (e.g. SEVCYMC for CytoTune 2.0, and SEVLMYC for CytoTune 2.1). All other assays are compatible with both kits.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
We do not have such primers for use in endpoint RT-PCR. However, we do offer validated TaqMan Gene Expression assays for each of the exogenous reprogramming genes (https://www.thermofisher.com/order/genome-database/browse/gene-expression/keyword/sendai+taqman). These assays are designed to specifically detect only expression of the exogenous genes found on the Sendai vectors, and will not cross-react with expression of the endogenous versions of these genes. Additionally, these assays can be used to detect the presence of each of the CytoTune Sendai vectors, for determination of clearance of the vectors from established iPSCs.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
For the Invitrogen CytoTune 2.0 kit, which contains 3 vectors (KOS, cMyc, Klf4), both the cMyc and the KOS vector can be cleared by a temperature shift to 39 degrees C. The temperature shift will be most effective for the cMyc vector, but should still work for the KOS vector.
Note: Before performing a temperature shift, first verify that the individual Klf4 vector has cleared. The Klf4 vector will not be cleared by a temperature shift, and performing the shift while the Klf4 is still present could cause problems.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
You can confirm the presence of the Sendai virus in your cells by several methods, including staining with anti-Sendai virus antibody, performing endpoint RT-PCR using the primer sets listed on page 50 in the CytoTune-iPS 2.0 Sendai Reprogramming Kit User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf), or real-time RT-PCR using the TaqMan Sendai Gene Expression assays, also listed on page 50 in the CytoTune-iPS 2.0 Sendai Reprogramming Kit User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
It can take as few as five or as many as fifteen passages for the virus to clear from the cell. In rare cases, Sendai sequences can persist indefinitely. Clearance rate is clone-dependent and can be confirmed by PCR or by anti-Sendai antibody. For more information about generating vector-free iPSCs, please refer to the user manual (http://tools.thermofisher.com/content/sfs/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
iPSCs must be monitored and growth medium must be replaced daily in order to maintain a healthy culture. In general, iPSC colonies should be passaged when the cells reach 70-80% confluence or when most of the colonies are larger than 700 µm. Please refer to the user manual for the full protocol (http://tools.thermofisher.com/content/sfs/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
We have used this combination of primary and secondary with no problems. IgG secondary antibodies will cross-react with IgM primary antibodies, since IgM share the same kappa light chains as IgG.
The IgG secondary antibodies that are listed in manuals are available, and they will work against IgG primary antibodies, as well as IgM primary antibodies, and others. For that reason, we typically have those IgG secondary antibodies on hand, and use them in most applications. That is the main reason it was recommended.
Please note that the Invitrogen CytoTune-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) has been discontinued.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The iPSC colonies can be easily visualized using alkaline phosphatase stain, such as the Alkaline Phosphatase Live Stain (Cat. No. A14353). In addition, reprogrammed colonies can be selected utilizing live staining with Tra1-60 or Tra1-81 antibodies that recognize undifferentiated iPSCs and enable the identification of reprogrammed cells from a variety of human cell types. Please refer to the user manual for the full protocol (http://tools.thermofisher.com/content/sfs/manuals/AP_Live_Stain_man.pdf).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The expected morphology of iPSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio. If you do not observe this morphology or the number of colonies observed is low, then the MOI used for transduction may need to be increased.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
We do not recommend using VPA with the Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit offers reprogramming efficiencies in the range of 0.02-1.2% with BJ fibroblasts. The reprogramming efficiency may vary for other cell types.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
You can cryopreserve iPSCs just as you would cryopreserve any pluripotent stem cells. Growth medium with 10% DMSO is recommended for freezing, or our PSC cryopreservation kit (Cat. No. A2644601) can be used
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
iPSC colonies will begin to form roughly 3 weeks post-transduction. Only one application of the vectors is required for successful reprogramming, enabling selection of iPSC colonies 21-28 days after transduction.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Avoid repeated freezing and thawing of the reprogramming vectors. Viral titers can decrease significantly with each freeze-thaw cycle and are not guaranteed for kits that have been refrozen or thawed.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The reprogrammed cells can be grown in standard iPSC culture medium. We recommend either Gibco KnockOut Serum Replacement (KSR)-supplemented medium in a feeder-dependent culture, or feeder-free in Gibco Essential 8 Medium. Please refer to the user manual (http://tools.thermofisher.com/content/sfs/manuals/cytotune_ips_2_0_sendai_reprog_kit_man.pdf) for the full protocol.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
If you want to reprogram PBMCs with Invitrogen CytoTune 2.0 under feeder-free conditions, you should follow the existing Invitrogen CytoTune 2.0 PBMC protocol, but plate onto Gibco Vitronectin or Geltrex matrix on day 3 instead of MEF, and then transition over to Gibco Essential 8 on Days 7-8 instead of Gibco KnockOut Serum Replacement-based PSC medium.
Efficiencies are typically lower than with feeder-dependent conditions, but you should still get some colonies.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
We recommend first to try increasing the MOI of hKlf4 only. For example: go from 5:5:3 to 5:5:6. If optimization is still required, then increase the MOI of KOS and hc-Myc. The ratio of KOS and hc-Myc must be 1 to 1, and the MOI of hKlf4 can be varied independently. For example: go from 5:5:3 to 10:10:3 or 10:10:6.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
An MOI of 5:5:3 (KOS, hc-Myc, hKlf4) is recommended for most cell types. The virus titer varies from lot to lot; the required volume for each MOI is listed on the Certificate of Analysis (CoA) for each lot of product. You may also choose to optimize your MOI as this may vary depending on the cell type. The ratio of KOS and hc-Myc must be 1 to 1, and the MOI of hKlf4 can be varied independently. For example: if KOS is 4, then hc-Myc must also be 4.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
MOI (multiplicity of infection) describes the ratio of viral particles to cells. The three vectors in the Invitrogen CytoTune 2.0 Kit should each be added to the cells based on an MOI recommendation. We recommend the following MOIs as a starting point, and adjustments can be performed if reprogramming efficiency is not optimal:
- Invitrogen CytoTune 2.0 KOS: Starting MOI of 5
- Invitrogen CytoTune 2.0 hc-Myc: Starting MOI of 5
- Invitrogen CytoTune 2.0 hKlf4: Starting MOI of 3
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
One kit (one tube of each vector, i.e., 3 tubes) is sufficient for a minimum of 5 wells of a 6-well dish at MOI of 5:5:3 (KOS, hc-Myc, hKlf4) with a recommended plating density of 2 x 10E5 to 3 x 10E5 cells/well for human dermal fibroblasts. The virus can only be used once, as viral titers decrease significantly with each freeze-thaw cycle.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
We recommend using the virus only once as viral titers decrease significantly with each freeze thaw cycle.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Yes, you must use all three reprogramming vectors together. Omitting one or two of the vectors will likely result in little or no reprogramming.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Upon receipt, these kits should be stored at -80 degrees C.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The virus does get passed on to daughter cells, and gradually over time its concentration diminishes. The virus can't leave the cells and infect new cells though, because the fusion gene has been deleted from the viral genome.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Although humans are not a natural host for SeV, and the virus is non-pathogenic to humans, appropriate care must be taken to prevent the potential mucosal exposure to the virus. The Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit must be used under Biosafety Level 2 (BL-2) containment with biological safety cabinet and laminar flow hood, and with appropriate personal safety equipment to prevent mucosal exposure/splash.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Yes, we have successfully induced iPSCs from mouse embryonic fibroblasts using our CytoTune-iPS 2.0 Sendai Reprogramming kit.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Yes. Please go here for a protocol (http://www.thermofisher.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols.html) or publication (http://www.thermofisher.com/us/en/home/life-science/stem-cell-research/stem-cell-research-learning-center/stem-cell-research-resource-library.html).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Our reprogramming kits have been validated for a wide variety of cell types, including human fibroblasts, CD34+ cord blood cells, and peripheral blood mononuclear cells (PBMCs). For a current list of publications citing the cell types validated using this method, go here (http://www.thermofisher.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/sendai-virus-reprogramming/cytotune-publications.html).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Integration-free reprogramming methods generate iPSCs that do not contain detectable vectors or transgenes. Traditional technologies used for reprogramming (e.g., lentivirus, retrovirus) integrate into the genome of the target cells. The resulting iPSCs and cells differentiated from those iPSCs will contain foreign DNA and could be unsafe and problematic for use in cell therapy and drug discovery applications. Furthermore, the integration could occur in a critical region of the genome, causing problems with unrelated developmental processes.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The Sendai virus vectors in the Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit are based on a modified, non-transmissible form of SeV, which has the fusion protein gene (F) deleted. The viral vectors maintain full infectivity to a wide range of cells; however they are no longer capable of producing infectious particles from infected cells because the viral genome lacks the F gene. The Sendai virus vectors contain transgenes that will express factors hOct3/4, hSox2, hKlf4, and hc-Myc. After transduction, the viral vectors will cause the cells to express these four genes, resulting in reprogramming
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Sendai virus, also known as Hemagglutinating Virus of Japan (HVJ), is a respiratory virus of mouse and rat first isolated in Sendai, Japan in the early 1950s. The virus is classified as mouse parainfluenza virus type I, belonging to the Paramyxoviridae family. SeV is an enveloped virus, 150-250 nm in diameter, whose genome is a single chain of (-) sense RNA (15,384 bases). The virus infects cells by attaching to the sialic acid receptor present on the surface of many different cells and is thus able to infect a wide range of cell types of various animal species.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Yes, the additional Klf4 vector allows the system to be fine-tuned by the user. The amount of Klf4 can be increased to enhance reprogramming efficiency, or decreased to minimize the total amount of virus.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The polycistronic configuration of KOS requires that additional polymerase activity be available to compensate for the combination of three genes on one vector. The Invitrogen CytoTune-iPS 2.0 system uses the extra polymerase from the hKlf4 vector to drive reprogramming in all vectors and enhance reprogramming efficiency. In addition, increased expression of hKlf4 also enhances reprogramming efficiency.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
KOS is an acronym for the genes hKlf4, hOct3/4, hSox2. This is a polycistronic vector, meaning all three of these genes are on one vector.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The Invitrogen CytoTune-iPS 2.0 Kit (Cat. Nos. A16517, A16518) offers higher reprogramming efficiency, faster clearance of the vectors, and lower cytotoxicity compared to the original Invitrogen CytoTune-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) that has been discontinued.
The 2.0 kit contains three vectors, one of which is a polycistronic vector (Invitrogen CytoTune 2.0 KOS), designed to deliver increased reprogramming efficiency. This polycistronic vector has a different backbone containing temperature-sensitive mutations in the polymerase-related genes, and this helps to clear the virus faster after reprogramming and causes less cytotoxicity to the cells.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The Invitrogen CytoTune -iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) is a non-integrating system that reprograms somatic cells into induced pluripotent stem cells (iPSCs). This kit utilizes four Sendai virus-based vectors, each capable of expressing one of the four Yamanaka factors: Oct3/4, Sox2, Klf4, and c-Myc. The expression of these transcription factors in somatic cells has been shown to be a critical factor in the successful generation of iPSCs.
Please note that the Invitrogen CytoTune-iPS Sendai Reprogramming Kit (Cat. Nos. A13780-01, A13780-02) has been discontinued and replaced by the Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Yes we did, and found that only the combination of KOS (3 in 1), cMyc, and Klf4-vector yields highest reprogramming efficiency. For instance, KOS and cMyc alone are not sufficient for reprogramming. Addition of Oct4 or Sox2 results only in a very few reprogrammed colonies. This is mostly due to an imbalance in the stoichiometry of the reprogramming factors, which may impair the reprogramming efficiency significantly.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit (Cat. Nos. A16517, A16518) is a non-integrating system that uses Sendai virus vectors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The Invitrogen CytoTune-iPS 2.0 Sendai Reprogramming Kit contains three Invitrogen CytoTune 2.0 reprogramming vectors, including the four Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. The expression of these transcription factors in somatic cells has been shown to be a critical factor in the successful generation of iPSCs. Only one application of the vectors is required for successful reprogramming.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
The full length Human FGF-basic (FGF-2/bFGF) (aa 1-155) Recombinant Protein is recommended for stem cells whereas the truncated variant, Human FGF-basic (FGF-2/bFGF) (aa 10-155) Recombinant Protein which is missing the first 9 amino acids, is recommended for use with neural and cardiac cells.
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.
Induced pluripotent stem cells (iPS or iPSCs) are pluripotent stem cells directly generated by introducing combination of genes coding for “reprogramming factors” into adult cells. These reprogramming factors include Oct4, Sox2, c-Myc, KLF4, NANOG, and LIN28. Yu, et al, generated iPS from a human mesenchymal cell line using lentiviral vectors carrying Oct4, Sox2, NANOG, and LIN28 genes (Science 318:1917 (2007)). Using a similar approach, Takahashi et al, generated iPS from human primary fibroblast cells by introducing genes coding for Oct3, Sox2, KLF4, and c-Myc into these cells (Cell 131:861 (2007)). iPS generated by reprogramming are similar to human ES cells in morphology, the capacity for unlimited proliferation, surface-antigen expression, gene expression, the ability to differentiate into cell types representing the three germ layers in vitro, and the ability to form teratomas after injection into SCID mice.
We have seen no effect on reprogramming efficiency as compared to Gibco Essential 8 Medium with Invitrogen CytoTune -iPS 2.0 Sendai Reprogramming Kit. We do recommend daily feeding during colony formation (days 7 - 28).
Find additional tips, troubleshooting help, and resources within our Cell Culture Support Center.