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I am staining thawed PBMCs with 5uM stock solution of CFSE for proliferation assay and also for control of the staining. However, in the results from Flow Cytometry of day 0 samples, (i.e. freshly stained and not cultured) there are 2 separated picks in lymphocyte population. Does anyone encounter this problem before and what could be the reasons.
And next question is that I tried to titrate the amount of CFSE using different volume of the same stock and I got sotr of concentration dependent proliferation inhibition: cells cultured with higher dose of dye showd less proliferation than those with less dye. Why this is so. The only reason I think could be the different size of cells, but I analyze only lymphocyte population, so this should not be the case.
I will appreciate any idea and advice.
What manufacturer's CFSE kit you are using? I think the Molecular Probes one we use has the stock concentration of 5 mM, of which we make a 1:5000 dilution to get 1 µM final concentration (and use 1 ml of that solution per 10^7 cells). Have you double-checked all your dilutions are ok?
I haven't had double peaks in freshly-stained lymphocytes as far as I can remember, but sometimes the freshly-stained samples seem to be too bright right after the staining, possibly due to small amounts of residual unwashed CFSE among the cells.
I think CFSE is somewhat toxic at high concentrations, so that might explain the lower proliferation rates you have seen - your "standard" concentration seems to be 5x higher than what we have found optimal for lymphocytes. Alternatively, since the cells are more thoroughly labelled, they may seem to proliferate slower, even if it is just the slower dilution of the dye you notice. Do you calculate the proliferation activity by counting cell divisions, or just the number of CFSElow cells? (Counting cell divisions should yield comparable results despite the dye intensity, but simply checking for the CFSElow cells is dependent on the initial staining intensity, at least to some degree.)
What do you mean by size affecting the outcome?
If you happen to have a dot plot or histogram depicting the situation, it might make troubleshooting easier. I have a feeling that perhaps I didn't quite understand the exact nature of your problem.
First of all I got 2 separate populations (2 peaks) in lymphocyte population when stained thawed PBMCs without incubation of cells (I used 5 mM stock solution for 1 million cells and incubate for 10 min according protocol C34554, Molecular Probes from Invitrogen). I expected not to have any additional population than one, as I do not think that 10 min incubation of new thawed PMBCs with CFSE is enough for them to divide. I used different volume of that stock for finding of the most optimum for my experiment (0.5ul; 1ul; 2ul and 3ul. See attached ppt: slide 1 and 2). I analysed them with FlowJo.
Next, I used from the same group of stained cells for 5 days incubation. I took 2 groups for each volume of CFSE mentioned above: irradiated cells (as negative control for proliferation) and non-irradiated cells. In non-irradiated cells I noticed concentration dependent inhibition of proliferation (see attached ppt slide 3). Does CFSE really have such inhibitory effect on the cells proliferation while being a marker for proliferation?
And finally, I decided to test different incubation time of the cells with CFSE.
I stained one million newly thawed PBMCs with 2 ul of 5 mM stock solution incubating for 10 and 20 minutes. Unstained cells were used as negative control. This experiment was done with PBMCs from 2 different donors. The cells were analysed by flow cytometry immediately (without giving them opportunity for dividion). However, still I got more than 1 peak (see attached ppt slide 5). As I understand different cells absorb CFSE in different amount (depending on size and surface), but I analysed in FlowJo only lymphocyte population. So it should not be the case.
P.S. I do not know how to attach file here. Could you please tell me about this. I am new in theis website.
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4 posts • Page 1 of 1
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