I am observing cell proliferation kinetics of B cells using CFSE staining. If I stain quiescent cells with CFSE and chase them for 3-6 days, they show distinct peaks for different generations. However, if I pulse already activated (but previously unstained) cells on day 3 or 6, I get a broad-spread peak moving over time, instead of distinct peaks. Is the multiple peak profile specific only for quiescent or synchronous cells?
Hi. Not sure if I understood your response. Actually, the problem is I am not seeing distinct peaks if I pulse dividing cells, while I can see multiple peaks if I pulse quiescent cells.
Dear Devi,
One possibility is that activated cells are blasted and they become bigger. When you label these cells they don't stain uniformly and as a result, you don't see distinct peaks.
On the other hand resting B cells have very uniform and unique size. Labeling these cells give unique peak. In subsequent divisions, CFSE signal dilutes to half.
You can easily verify this phenomenon by looking the cells under a microscope.
Hope this helps.
Mrinmoy
Mrinmoy,
Thank you for the suggestion. It is true that the cells bet bigger after activation. Considering a particular case I have tested-
I stained cells on day 0 and observed them till day 6. Distinct peaks could be seen from day 2 to day 6. On the other hand, I took unstained activated cells on day 3 and stained them. I could not see distinct peaks from days 4, 5, 6.
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