Hi!
I want to make conditioned media from BMSCs in order to study their paracrine activity in other type of cells. I made the CM when BMSCs were at 90% confluence. Simply, I aspirated basal media (DMEM 1X, 15% FBS, 1% P/S, 1%NEAA, 1%L-Glu), washed 3 times with PBS and incubated the cells only with DMEM 1X for 24 hours at 37ºC. Next day, I collected supernatant, centrifuged it (to eliminate any cellular debris) and stored at -80ºC.
The question is... the cells which have been conditioned, can be used for do more aliquots of CM? I saw that in 24 hours many of them died, so I subcultured them in a shorter flask (from T75 to T25) and now, most of them look well but there are some that seem "damaged" as they were "stressed out" to make CM.
Looking forward to advices!
Many thanks,
Claudia.
Subhash C. Juneja my exact question is if cells which I've incubated in DMEM 1X for 24 hours could be used for future incubations with DMEM 1X (to collect more aliquots of CM).
I'm not familiarized with conditioning cells and I don't know if this cells, since they've been stressed in conditioning (they were 24 hours with depletion of nutrients that normally have), could give rise to erroneous results. I know that CM is a "black box" and it's difficult to obtain 100% reproducibility but I have doubts about the remaining cells.
Sorry if my question is not well understood.
Hi, Claudia.
In teory, CM produced will differ depending on the state of the cell. So if you want to produce "good" CM, it must be produced from healthy cells, not from stressed cells
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