Dear community,
i'm looking for a possibility to determine the confluency of granulosa cells in a dish. We stimulated cells with Pam3Cys-Ser-(Lys)4 (a TLR2 agonist) and observed them under the microscope. Unfortunately the stimulant leads to a precipitate in our DMEM-F12 medium, which makes it hard for me to determine the confluency via ImageJ. You can find a picture attached
Does anybody have an idea how i could solve the problem?
It would be great if you help me with the confluency OR the precipitate!
Thank you very much!
Hi
I am not sure that what you are looking at is a precipitation of your TLR2 ligand. I've used FSL-1 and PGN before (in other cell lines) and never saw precipitation. To me your cells are reacting strongly to the ligand. I've never seen granulosa in culture but the bright round cells look like apoptotic bodies and maybe the precipitates are cell debris. Have you adjusted the concentration of your ligand to avoid toxicity?
Good luck
Thank you for your answer!
I tried lower concentrations of Pam3 which led to no precipitate. In this picture is a lot of apoptosis but i also get the "precipitate" without cells in pure DMEM F12 medium. Further i've done a couple of LDH-Cytotoxicity Assays, which showed that Pam3 doesn't cause cell death, significantly.
Any other ideas?
- Badges
- Science method