could anyone please help me figure out what I could be doing wrong ?

how could the untreated group show higher ROS than LPS grp ?

I am using primary astrocytes , treating with 1 micogram per ml LPS for 24 hours

for the flow cytometry , I incubate with mitosox red for 30 mins , trypsinize the cells and centrifuge them then resuspend the pellet in facs buffer

(to avoid losing the pellet , I sumetimes leave a small amount of medium surrounding the pellet before I add the facs buffer .. could this be an issue ?

Thanks :)