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I am trying to assess my cell proliferation by using a CFSE assay, however I can't obtain more than one single pic on the flow cytometer after 5 days, even if I know that my cells did proliferate.
Does anyone have an idea about it?
Thanks a lot
The most obvious reason is that there's something wrong in the CFSE staining you've done. Double-check your protocol that you did all correctly. If I recall correctly, CFSE is very sensitive to light, so you must protect it or else some few hours exposure can spoil it.
Btw, what methods you use to ensure that your cells have prolified? Do you just check them under a microscope or count them in a chamber, or just look at the colour of your medium?
What cells do you use? For example T cells need proper stimulants to make them divide (e.g. PHA if you want unspecific proliferation), as well as the feeder cells. It is easy to think your cells proliferate, when the actual metabolism and medium colour change is caused by the feeder cells, not the cells you want to study. (And you cannot count the T cells directly because of the feeders)
You could provide me some more information about your cells and the protocol, and I can try to come up with alternative solutions.
Thanks a lot for your reply.
I am using primary T-cells indeed, but I tried with some MM6 macrophages and K562 (to check if the cell type might make a difference). I am stimulating my T-cells with PMA instead of PHA, but as these twomitogens are pretty similar, I don't think this make a huge difference.
I am counting my cells on a Neubauer chamber with trypan blue, and I am also using a nucleocounter to get the number of cells/their viability.
I wasn't aware of the CFSE light sensitivity. I will protect it from the light from now.
About the CFSE concentrations, I diluted it quite a lot (down to 500 times the lowest concentrations proposed by the manufacturer) and also used it highly concentrated, but it doesn't make any difference (well apart from the GMFI of my single pic obviously).
If you have any ideas, please let me know, because I really have no other clue.
P.S: I bought two kits as I was supposed to use it quite a lot, and I have the same results with both batches
This is Avinash, PhD student at QUT, Australia. I have been doing PBMCs proliferation using CFSE. I stain my cells with 5uM CFSE (5mM stock). In my unstimulated control cells, I cannot see any proliferation under microscope when compared to my antigen stimulated and ConA stimulated wells. Even there is color change between unstimulated and stimulated cells.
However, when I analyze them on CFSE, I get more than one peak in my unstimulated histogram. My ConA looks very good (with 4 peaks minimum). But there is not much difference between my vaccinated antigen stimulated and unstimulated ones.
Could anyone please help out with this.
I do a proliferation assay after pulsing PBMCS with CFSE (5micromol for 2-10million cells) and then staining with CD3. I sort the cells and then stimulate them for proliferation with PHA(2microlitre). I also stain the cells with CD4 and CD8 on the day of analysis. I add PI jus before running the cells on the flow cytometer to discriminate for live vs dead cells.I am worried because I only get a single peak for each day (1-5 days expt). am i using too much CFSE? is that making it difficult to discern the peaks? Can somebody help with the trouble shooting. I also apply single compensation controls for the first day and find no spill for the rest of the days by using the same settings.
Thanks in advance
How does the single peak relate to your negative control? Do all your cells have full or lowered CFSE intensity? Does the intensity go down each day?
Having too much CFSE should not be the cause, because even high concentrations diminish quickly due to the cell divisions. In my experience, high CFSE concentrations easily shoot the cells off the axis but does not affect the peaks once the cells start to divide - except that their intensity is high in general. Rather, you might have too much PHA. It is a very powerful mitogen and chances are that it makes all the cells divide -> all cells have undergone equal amounts of cell divisions -> one peak.
You could try to control this by comparing your unstained cells, fresh CFSE stain and one sample from, say, day 4 or 5. If the day 4-5 sample is between the two others, it most likely indicates that the case is like I mentioned above.
I typically use 1 µl/ml of PHA if I just want the T cells to multiply - even that, I think, is plenty. For CFSE staining I may use even less (0.5 µl/ml or so) in order to be able to discriminate each cell division (=peaks) properly.
Thanks a lot for your reply
I did MLR proliferation assay with 10micromole of CFSE and did not see any division till day 5 but there was a slight movement in the original peak.
On day 8 i saw a popultion which was completely negative for the CFSE and also one small peak which was slightly positive for CFSE but the cells were dead almost should i consider as divided cells.
Please help me regarding the post analysis of cfse proliferation, Do I have to use any special softwares for that? or any gating strategy can be used? On day 8 I get some extended population from the original population, but unable to differentiate them as individual cell divisions and finally gating them becomes impossible. Please help me regarding the gating strategies that can be used.
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