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how to quiesce human PBMCs without them sticking - (Jun/22/2011 )

Hi, I would like to quiesce human pbmcs (particularly the B cells) for 2h following separation from blood using Histopaque-1077. Unfortuantely when I tried putting the pbmcs into a culture flask and incubating them for 2h, the majority of cells stuck down and I couldn't remove them (for flow cytomtry). Anyone have any ideas why the cells stuck and how to remedy this problem so I can culture my cells of interest (B lymphocytes) and harvest them for subsequent analysis?



If you put unsorted PBMNCs in a culture flask, to my knowledge the first cells becoming adherent are dendritic cells (a way to remove them from PBMNCs). How do your stuck cells look like under the microscope?
Why don't you just keep your cells cooled on ice before FACS?

Best, M