Splenic adherent cells wont adhere!
 

Hi, all
I am newbie to immunology and trying to culture mouse spleen adherent cells to study cytokine production upon LPS stimulation.

I dissect spleen, mechanically dissociate using the end of plunger, and plate onto petridish/culture dish in RPMI1640/Heat Inactivated 10% New born Calf Serum/Pen/Strep for one hour.

After one hour incubation, I can see lots of cells adhere. :happy: So I wash 3 times to remove non-adherent cells and incubate overnight. Then for some reason almost all cells come off the plate and not much left on the plate!
:pissed:

Please Immune expert here could you provide any solution/mistake I am making?
Thank you veery much in advance!

yzf