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 ??
if anyone has some ideas ....
Submit your paper to J Biol Methods today!
bone marrow derived macrophage
No replies to this topic