I am conducting stimulation assays with mouse splenocytes and after 60 hours, when I try to collect them for Flow analysis, I find very few cells in my supernatant. I have observed that the cells form a layer on the base of the wells of 24 well plate. Should I use trypsin or scrap them to collect it? I also know that the mouse splenocyte single cell is in suspension. If using trypsin, also please detail the process.
I would appreciate your views.
Trypsin can be used at a lower concentration followed by scrapping..
Thank you for the reply. Can you elaborate on the quantity and method?
hi
did you remove the spleen infiltrating macrophages during splenocyte isolation?? i think the cells adhering are not splenocytes (B/T lymphocytes) but actually macrophages. You can remove them by simple plastic adherance.
Generally when you get splenocytes, you are taking ALL the cells from the spleen (lymphocytes, red blood cells, fibroblasts, macrophages, etc) even if you lysed RBC you still have all other cells.
Probably your attached cells are all others I have just mentioned and the suspended cells your lymphocytes.
Good luck.
Generally most of the cells adhere. trypsinization and scrapping can help you priyanka