How to isolate Macrophages from mouse spleen and liver - for downstream functional assays by FACS (Apr/29/2008 )
I am new to macrophages. I know how to get them out of peritoneum. But, am looking for a good way of purifying them from spleen and liver (Kupffer cells) to do some phagocytic, apoptotic assays by FACS. Anyone? Someone suggested using Miltenyi CD11b beads. Does that affect the function?
If plating is the best way to purify them (specially from IHL), whats the best way to get them off the plate? Lidocaine? EDTA? Anything else? I can't use trypsin because I need to do FACS staining. What kind of viability do you see with your favorite method?
Also, how long do I need to plate them for before they will all stick to get rid of contaminating cells.
Is plating essential for their function? Can I do say, the phagocytosis in suspension culture (essentially in a FACS tube that gets rocked/mixed every few minutes) if it is a short term reaction? Also, how fast do they stick to the plate/FACS tube?
Any links/references for detailed protocol will be highly appreciated too. I know very few basic things.. not much.
thx a ton
Someone suggested using Miltenyi CD11b beads. Does that affect the function? These work fine for isolation of monocytes from the splenic tissue and their function is normal.
I have used the plating method to remove "contaminating" monocytes. We would leave the suspension for 2-3h but others have left overnight. To remove them from the plastic, I have used straight pipetting force for RAW cells and if too sticky, 0.53 mM EDTA in PBS works ok.
I use many monocytic suspension cultures for cytokine assays and do not differentiate them (where they will adhere) in order to allow easier analysis by flow cytometry.
Hope this helps,