Hi Sangeen Khan . The cells look dead or dying, which might explain why you are not seeing a pellet. It is likely that you have some contamination problem going on if the media is cloudy after 24 hr incubation. Try thawing out a new batch of cells and use new fresh media.

Hi Sangeen Khan, I agree with Stefansson, your cell or media is somehow contaminated. Most likely mycoplasma, but can be just other bacterial contamination too. Also, try thawing a different vial of cells from a different batch and use totally new media other than the batch you prepared already. If you still get a contamination problem, then you can test for mycoplasma contamination, or use an anti-mycoplasma drug, for example, Normocin.

thank you so much for your recommendations @Stefansson and @Khan

You can check with the dye WST-1 to measure the cells are dead or not. The cells look sick.

For refs for the WST-1 method, please looks at:

Article Multiplex stress resistance in cells from long-lived dwarf mice

Article Fibroblast cell lines from young adult mice of long-lived mu...