Hi everyone. Hoping someone could shed some light....
I've been having real issues with staining murine bone marrow DCs positive for CD11c and MHCII. Currently about 1% of the non-adherent cells I collect are staining on flow cytometry as CD11c+ and about 30-40% as MHCII +. I have no idea what I'm doing wrong as morphologically they look like dendritic cells!
My protocol is as follows:
I culture about 15-20 million murine (BL6) bone marrow cells in T75 flasks for 6 days and run FACS on day 7.
My culture media includes:
20ng/ml of rGM-CSF and 5ng/ml of IL-4
in 10% FCS, 100 U/mL pen and 100ug mL strep
in RPMI 1640.
(on day 3 I add another 10 ml of media with 20ng.mL GM-CSF and 5ng/mL IL4)
I collect non adherent cells on day 6 and spin down at 1800 rpm for 7 mins at 4 degrees then resuspend in 1%BSA-PBS. I stain cells with the appropriate MHCII and cd11c antibodies ( in 96 well plates 100ul of cells per well (polypropylene plates)) for 20 mins and spin this down 1800 5 min 4 degrees, remove supernatent and resuspend in 4% PFA. I filter these through a cell strainer and then top up with 100ul PBS. I then run these samples (in polypropylene tubes).
Does my method sound like it could be causing the problem? As I said, morphologically the cells look like DCs- I have been advised to look for clumps of cells that look quite spiky! I'm so certain its the staining that's not working and not a culture problem. Any suggestions would be very much appreciated!