我正在使用你们推出的FreeStyle Max 293表达系统，细胞活力至多达到87–90%。这正常吗？你们能否就提升细胞活力提供一些建议？我正在使用125 mL和250 mL带透气盖的非挡板式聚碳酸酯振荡培养瓶。使用挡板式培养瓶会有帮助吗？我应在培养基中添加Pluronic试剂吗？
培养用于转染的细胞应保持起始细胞活力在90%以上。转染后，细胞活力会在3-6天后缓慢降至90%到80%区间，7天乃至更长时间后发生更显著的下降。这是使用我们推荐的实验方案时的情况，该方案不推荐在转染后对培养基进行换液或补充。
我们建议用户对一些参数进行检查，以确保获得高细胞活力：pH在7.0左右，无可见的细胞团块，CO2水平在7–8%左右（需对该值进行调节以达到适当的pH值），培养体积应为培养瓶标称容积的40%或以下，培养箱温度为37°C。如果您的培养箱中配有振荡平台，则振荡台产生的热量可能足以升高培养箱温度。我们推荐在振荡台上贴一个温度计。如果怀疑培养物的氧气供应不够充分，则使用带挡板的培养瓶可能会有帮助。光照也可能会造成某些培养基成份的降解，我们建议妥善处理和保存培养基。如果您正在使用FreeStyle 293表达培养基，则无需添加Pluronic试剂。
注意：细胞聚团的两个主要原因为：1）细胞培养密度过大，和/或2）振荡不充分。因此，充分的搅拌，按照细胞传代时间表规律的传代，并以推荐的密度培养细胞就能够防止细胞聚团。如果发现细胞聚团情况，我们则建议放弃培养物，另取一管新的冻存细胞重新开始培养。用户可能需要在一段时间内每次传代时对细胞剧烈涡旋10-30秒，直至大部分细胞都以单个的形式存在。此外，用户可于每次传代时通过沉降的方式来清除聚团细胞。用户可增加摇床的转速，并确保每次在细胞不超过1.5–2 x 106个细胞/毫升的密度时进行传代。只要细胞活力保持在95%以上时，可增加摇床的转速。在细胞转染后的至少初始2-4小时降低转速，以确保DNA-转染试剂复合物能够良好贴附和进入细胞内。用户也可在最初两次传代时以1:1000（1:200最高）的稀释比例添加抗结团试剂（货号 0010057AE），之后在转染前在不含抗结团试剂的条件下进行传代。请勿在转染过程中的任何时间点添加抗结团试剂，因为该试剂会对转染过程造成干扰。不过，转染数小时之后即可加入抗结团试剂。
FreeStyle Max 293表达系统与FreeStyle 293表达系统之间的主要区别是什么？
除转染试剂和用于配制转染复合物的培养基不同之外，这两个系统拥有相同的组分。FreeStyle Max 293表达系统包含了FreeStyle Max试剂和OptiPRO SFM（非动物源性的无血清培养基），而FreeStyle 293表达系统则包含293fectin转染试剂和Opti-MEM I减血清培养基（无血清培养基，但不是非动物源性）。
I am using your FreeStyle Max 293 Expression System and my cell viability is at most 87-90%. Is this normal? Do you have any tips to increase the cell viability? I am using 125 mL and 250 mL non-baffled, polycarbonate shaker flasks with vented caps. Will using baffled flasks help? Should I add Pluronic reagent to the medium?
Culture viability should be in the high 90% range for the seed stock that you are maintaining and growing for the transfections. After transfection, the viability will slowly decrease to the lower 90% to 80% range by about 3-6 days post-transfection and can drop more dramatically at 7 days or longer. This is when using our recommended protocol, which does not suggest any media changes or supplementation post-transfection.
A few parameters we recommend checking to assure high culture viability are: pH at ˜7.0, no visible clumping of the cells, CO2 levels at approximately 7-8% (this should be adjusted to achieve the appropriate pH), volume of culture at 40% or less of nominal flask capacity, and incubator temperature at 37 degrees C. If you have a shaker platform setup in your incubator, the platform may generate sufficient heat to increase the temperature of the incubator. We recommend using a thermometer attached to the shaker platform. Use of baffled flasks may help, if insufficient oxygenation of the cultures is suspected. We also recommend handling and storing the culture media properly, as light exposure can result in breakdown of certain media components. If you are using FreeStyle 293 Expression Medium, supplementing with Pluronic reagent is not necessary.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
What is the main difference between the FreeStyle Max 293 Expression System and the FreeStyle 293 Expression System?
The two systems have the same components except for the transfection reagent and the medium supplied for formation of transfection complexes. The FreeStyle Max 293 Expression System contains FreeStyle Max Reagent and OptiPRO SFM (serum-free medium that is devoid of animal-origin components), whereas the FreeStyle 293 Expression System contains 293fectin Transfection Reagent and Opti-MEM I Reduced Serum Medium (serum-free medium but not animal-origin free).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Which vectors do you recommend for use with FreeStyle system?
Generally, any mammalian expression vector should be compatible with the FreeStyle expression system. Some of the more popular choices include pcDNA3.3-TOPO TA vector (Cat. No. K8300-01), pcDNA3.4-TOPO TA vector (Cat. no. A14697), pcDNA3.2 DEST vector (Cat. No. 12489018), pCEP vector (Cat. No. V044-50), or pcDNA4/HisMax vector (Cat. No. K864-20).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
