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Dying Monocytes - Problem with cell culture of monocytes (May/13/2009 )

Hello everybody,
Unfortunately I have problems with mouse monocytes:
I separate them from red blood cells and granulocytes with histopaque-1077. After this step, I transfer the “interphase” (leukocytes and monocytes) into a fresh tube and wash it two times with culture medium (RPMI + 10% FCS). Then I transfer the cells into a six-well-plate in RPMI + 50% FCS and let them sink down and adhere for an hour. After this hour, medium and non-adherent cells are removed and culture medium (10% FCS) is added.
They are dead by first or second day after preparation.
I presumed that RPMI + 10% FCS; 37°C; 5% CO2 would be good conditions. May it be that the culture plate must be coated with poly-L-lysine or something else? A lab-mate told me that this is not necessary but I know others coat their plates.

(Yes, I used the search function but this problem does not seem to be covered in other monocyte discussions ;-)

-Zorndeig-

Zorndeig,

When you aspirate to remove non-adherent cells, don't aspirate all medium completely and leave some behind to keep the cells "wet". See if this works.

Jay
CellSpecific
http://www.cellspecific.com

-Jay Dela Cruz PhD-