I am trying to differentiate microglia following the simiplied protocol from the Blurton-Jones lab (Article Development and validation of a simplified method to generat...
).On the second step of the protocol (HPCs > Microglia), I collected the non-adherent cells on Day 10 and plated them onto matrigel coated wells with iPS-microglia media (DMEM/F12 + Insulin-transferrin-selenite + B27 + N2 + glutamax + NEAA + monothioglycerol + insulin) and put it in the incubator (37C+ 5% CO2) overnight. The next day, the wells were all cloudy with precipitate and had congealed into a jelly like substance on the bottom. It is most likely not bacterial or fungal contamination because the color is unlike a normal contamination and also I could not see anything moving in the wells. To test if it was the media, I put just the iPS-microglia media in an empty 6-well plate in the incubator overnight and saw the same cloudy media with congealed bottom again. I believe it's something in the media that is reacting upon heat or humidity that is causing this chemical reaction. Does anyone else have experience with this or have any suggestions?
you said that you use non-adherent cells. These cells tend to suspend in your media and they probably make a network within your media and make that cloudy jelly-like form. I propose using stirring culture vessels, to avoid this. Also, you can try using a higher amount of culture media.
I agree with Sravan Goparaju , cloudy and jelly sounds like a coating problem. Were you using freshly prepared Matrigel coating plate?
By the way, irrelevant to this discussion, how was your experience with STEMdiff Hematopoietic Kit? In my hand I got high percent of dead cells when harvesting the Supernatant, as dead cells also float. Anyone has suggestions? Thanks!
Unfortunately, I did not work in this field. My wishes for you to find a solution to your problem as soon as possible.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts