想问下大家，用no feeder体系诱导ips.细胞大量死亡

lchanlon

I think what happened in your iPSc generation experiment is normal. This is happened in my experiment. But I am a little bit confused about 100, 000 cells per plate/dish. What kind of dish are you using for lentivirus transduction? if 100, 000 fibroblast cells was in one well of six-well plate, this might be good. It is terrible in a 100-mm dish. Based on my experience on human primary fibroblast culture, cell density is a factor to keep primary cells healthy. Are you sure the polybrene working concentration is 5 ug / ul? I remember I was using 6 ug / ml for virus transduction. After cells reseeding, what i was doing is culture the cells for 2-3 days in fibroblast medium, then replace medium with tesr1 or E8. Cells is not happy after media changing and many cells are dying. The lentivirus for transduction is concentrated virus or just cell supernatant. is it commercial virus? if not, what kind of method did you use for virus packaging? Using lipofectamin or calcium?