After using flow cytometry to check early activation of Jurkat cells by estimating CD69 expression after 4 hours of incubation with CD3/CD28 ABs, I need to do the same for the late activation. So I tried to incubate them with CD3/CD28 ABs (together) for 24 and 48 hours and I didn't get any CD25 expression by flow cytometry. Should I change the protocol? Maybe add IL-2 to the medium used for incubation with CD3/CD28? Or just try 72 hours? Thank you for any suggestions!