Macrophage flow cytometry - (Sep/24/2007 )
hello, all
i'm having some problems with my macrophages...i use PMA differentiated THP1 human monocyte culture. the cells differentiate fine, but when i try to apply my usual staining protocol, i somehow loose the cells!
i have several questions regarding this:
1) is it safe to scrap the cells with a plastic scraper (are macrophages too sensitive for that??). I cannot trypsinize them because i stain for surface markers and am afraid they will be destroyed by tripsinization.
2) the second problem i've had is that i somehow can't pellet my cells. i've tried to spin them at 4min/200G, 4'/600G and 10'/600G but nothing seems to work...
3) when i put the unstained samples in my cytometer there are very few events where there should be thousands! could it be that 200G is to much for mycrophages to survive??
does anybody have a macrophage staining protocol to share?
thank you all for any help!!
ruđer
i'm having some problems with my macrophages...i use PMA differentiated THP1 human monocyte culture. the cells differentiate fine, but when i try to apply my usual staining protocol, i somehow loose the cells!
i have several questions regarding this:
1) is it safe to scrap the cells with a plastic scraper (are macrophages too sensitive for that??). I cannot trypsinize them because i stain for surface markers and am afraid they will be destroyed by tripsinization.
2) the second problem i've had is that i somehow can't pellet my cells. i've tried to spin them at 4min/200G, 4'/600G and 10'/600G but nothing seems to work...
3) when i put the unstained samples in my cytometer there are very few events where there should be thousands! could it be that 200G is to much for mycrophages to survive??
does anybody have a macrophage staining protocol to share?
thank you all for any help!!
ruđer
Hi, never worked with THP1, but for my bone-marrow derived macrophages i use PBS-3mM EDTA-10mM Glucose to detach them, not as harsh as trypsin. also i spin them down for 10 min at 1200rpm = 311g, so i dont think you'll be killing them at 200g. Do you actually see a pellet?
other than that, i follow standard staining protocols. my usual problem is that macrophages are quite autofluorescent so compensation gets tricky.
Good Luck!