Hi, I wanna trunsduce iPSCs with lentivirus for differentiation.
There are some problems. I grow them on MEF, and I think this feeder will become trunsduced more than my colons. And another question, I grow iPSCs with KSR, but my viruses have been prepared by FBS, and I am working with fresh virus, how should I manage this? Can I remove KSR completely and work with FBS during differentiation process?
another dillema, how much this colons will become trunsduced?especially their centers!! If I work with EB, or EB single cells, or even tiny colons of iPSCs, would it be ok?
Can anyone kindly give any suggestions or comments?
Thank you,
Hi,
Try to grow iPSC cells without MEF cells. Cover the plate with Matrigel solution. Use a MEF conditioned medium. Optimize the amount of Activin A in MEF conditioned medium. Passage hiPSC as a single cells by Tryple. Try to transduce hiPSC in suspenction (in eppendorf) before or 3 hours after they attach in to the plate covered with Matrigel solution. ROCK inhibitor should be add to the medium after transduction or/and passage hiPSCs as a single cells to improve their viability.
Prepare clonal selection after lentiviral transduction.
Knockout serum replacement (KSR) is an alternative for the Fetal Bovine Serum (FBS), so they can be used interchangeably.
Good luck!
thank you Jastyna. If you please, I wanna think about your suggestion. then ask a few more question.
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