Hello everyone
I am trying to evaluate IFN-g secreting CD4+ T cells in healthy individuals by flow cytometry. The following protocol is what i use for stimulation and staining:
1. A total of 1 million PBMCs are stimulated in RPMI with PMA/Ionomycin (cell activation cocktail, biolegend) in the presence of Brefeldin A for 6 hours.
2. Then cells are washed 2 times with staining buffer (PBS+ 2% FBS)
3. Washed cells are fixed with Paraformaldehyde 1% solution for 20 min in 4C
4. Then they are washed 2 times with saponin solution
5. Antibody (PE IFN-g) is then add to cells in the presence of saponin for 30 min in 4C
6. Again 2 times wash with saponin
7. Read with 3 colors flow cytometer FACS Canto
At the beginning there was no problem, but now the whole cell populations goes above gating lines with the same condition!! Does anybody has a clue? The attached files show situation.
I am not entirely sure if I understand you correctly. However, the first thing that I am noticing is that the population you describe is going off scale. As far as I can see this seems to be due to overcompensation. You should be able to check if this is the issue.
Johan
Thanks Johan Teunis
Actually both population were read with same compensation and I didn’t change the setting.
Hi Yashgin,
I agree with Johan. Both of your plots are overcompensated. This is why you see a red line along the y-axis where the positive population is falling off scale.
Are these 2 plots from different donors? You may be seeing donor to donor variability in IFNg production.
Nicole.
Thanks Nicole Acuff
Yes the plots are from different donors. You mean that overcompensation could lift up negative population?
The second population is from a healthy donor. Is it possible that a person be 100% positive for IFNg?
Adjusting the compensation should not move your negative population, it will just bring the IFNg+ cells back on scale. How many colors are in this panel? If there is a large compensation error, it could be that one of your other markers is leaning into the IFNg channel giving you a false positive. Or this donor could have recently been ill and so has a greater frequency of circulating IFNg+ cells.
In the second plot, i used three colors, but i don't think this is the problem. Because i tried it many times in single color and also double colors, got same result. Moreover, i examined different donors too (patient and healthy). In patients (kidney transplant), the negative population was at the right place (lower left). In healthy ones, i didn't get proper results.
I attached another file which shows result after changing compensation and channel voltage in three colors sample. some parts of the negative population comes down but you are no longer able to see anything in unstain tube!
Is it possible that something affect cells during stimulation process?
ah, now I understand your question. The negative population moves upwards. This is most likely due to background signals or unspecific binding of antibody. However, if you are using similar concentrations of cells with similar concentrations of antibody this should not happen. What you should do is look at the unstained sample of both donors to make sure the autofluorescent signal of the cells are different. If so, you should gate based on the donors own negative population. Otherwise we need more information of the entire experiment.
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