I am trying to identify the different cell populations in spleen from C57 mice.
It is the first time I am using a cytometer and nobody in the lab is able to help me.
I count the white cells with the cytmter after lysis of the red cells and it is OK. Then I use the FCblock, the antibodies, do the washes...
the problem is when I go back to the cytometer, I don't have cells in my gate of interest anymore (less than 20%), and the rest are events with a very high side scatter and a very low forward scatter (this means that all the cells are stuck together).
I use a solution of PBS (free of CaCl2 and MgCl2, from invitrogen) to which SVF and azide was added to reach 2% SVF and 0,16% azide.
Can this be the reason why my cells stick to each other ?
Does anybody can tell me what kind of solution I should use ?
thanks a lot
I don't manage to measure ROS production from bone marrow derived macrophages, neither in cytometer (DHE and DCFHDA) nor with the NBT reduction method.
I tried by stimulating the cells with IFNg (20 U/mL) and LPS (1 microg/mL) overnight and 30 minutes to 1 hour of DHE or DCFDA or NBT.
I also tried by treating the cells with PMA (20nM and 200nM) directly in DHE DCFDA or NBT :
- after IFNg and LPS stimulation overnight
- without any pre-treatement
I checked my cell population and saw that 90-95% of the cells are cd11b positive.
this means that :
-either the cells are not stimulated enough (differentiated enough) ??
-either the cells are "tired" and not able anymore to produce ROS ??