Hello researchers,
I am currently working on cell proliferation assays with CD8+ T-Lymphocytes which I extract from the mice spleen. I label CD8+ lymhpcytes with CFSE and stimulate them for 2 days with Anti-CD3/Anti CD-28 plus IL2. I monitor cell proliferation peaks on days 0,2 and 3 using FACS.
I analysed the data with FlowJo proliferation modeling. In Figure 1, you see the day0: fresh extracted cells with CFSE-label which are undivided.
In Figure 2, you can see day 2. The initial peak has shifted one log to the left and there are 3 peaks now. Percent divided is approx. 70% whereas proliferation index is 1,01.
My questions are as follows: Is it really possible to have 70% divided cells on day 2 that have the proliferation index of 1,01? or Do you think that the peak zero (small peak on the right side) is just an artefact which confuses the FlowJo and the undivided peak is the middle, greater peak?
If you think that peak zero (small peak on the right side) is an artefact, what might be the reason that a mini pre-undivided peak emerges on day 2?
Many thanks!!!
Hi Defne,
Personally, I am having similar problem, and unlike you i dont have double peak, but one peak that always shift left.. for me i think its bleaching, rather than the dye dilution due to cell division.
For your data, I think may be you take peak 0 in your first picture as undivided cell.. again i am still in doubt, as i couldnt find explanation for the shift.Let me know if you come up with something.
One way for solve this issue is to have a control well in which CD8 T cell CSFE labelled are left without any stimuli, the reason is that CFSE dye (like all proliferation dye) are often use in great excess, they became fluorescent when they bind to protein ammine group but over time the protein composition of the cell change and the zero peak on day 0 is not the same zero peak at day 2 so in order to have a correct interpretation you must set the peak of undivided cell using un-stimulated cell at day 2, not at day 0. The analysis on day 0 must be done to be sure the staining is homogeneous, not for set the undivided peak.
I agree with Dario. Keep some unstimulated labelled cells to acquire at day 2. This is to my mind the best control and we always did so as well.
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