Hello, everyone
Do you know some techniques to improve the efficiency of reprogramming if I use a kit (cytotune-ips 2.0 sendai vector) expired in July 2021?
Thank you for your answers!
Have you tried this expired kit before? Do you know how many freeze-thaw cycles the main stock has gone through? If used previously, do you know whether the vector has sufficient aliquots for reprogramming a single cell line at a time? (though CytoTune strictly prohibits making aliquots, there are some studies recommending it) How stable the temperature was in the ultrafreezer that you guys kept the 3 Sendai vials?
Depending on your answers, I would definitely recommend you to use a MOI of 10:10:6 , especially if you are reprogramming some freshly harvested primary tissues (i.e. fibroblasts), which resists more to the cell death induced by increased MOI to my experience. If you think using a double-MOI would be a huge waste, then go with the ORIGINAL 5:5:3 for 300.000 cells at a time, and check the day 3 post-transduction controls for the abundance of Sendai vectors by RT-PCR. Which would tell you so much about the stability of vectors in each vial prior to the emergence of the first colonies, and you can even increase the MOI for a certain vector (i.e. if you detect no or very less KOS vector, you can adjust it's MOI empirically)
Next, follow the CytoTune manual strictly and keep viral vectors on ice all the time, after partially thawing them quickly in waterbath.
Make sure to perform cell counting and stuff (we usually get cells to be transduced in 300 uL final volume per transduction) before removing virus from -80. This is the most important part, as cells may resist being in solution for a brief 10 minutes time, but Sendai virus cannot. It has to be immediately used upon thawing by centrifugal transduction as recommended by the manufactuter.
Try not to transduce less than 300.000 cells per transduction and make sure the kit (if used previously) has enough vectors remaining in each 3 vials for your reprogramming experiment. Lastly, make sure to conduct centrifugation in a round-bottom centrifuge tube. Best of luck!
You're welcome. One last thing, I would go for 5:5:3 for 100.000 at one time (assuming you have sufficient primary somatic cells to try again), and check for the abundance of each vectors at day 3 post-transduction by RT-PCR on the agarose gel. If there is suspiciously low yield in any one of the vectors (which happens to be the case as we experienced significant decrease in KOS vector only, and increased it's individual MOI for the following reprogramming), try increasing its MOI in the next round!
I will keep it in mind for the next test. If you don't have any inconvenience, can I send you a private message? I have some additional questions
Thank you! Onur Can Begentas
I really appreciate it
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