To check the lymphocyte proliferation, I separated PBMC from human blood and incubated them with CFSE. Then I check the FITC immediately. The FCM show two clusters in the FITC, why the cell get the CFSE different? Secondly, I incubated PBMC in dish 2 hours to purify the lymphocytes before CFSE treatment. There are two clusters still. Why is that?
Hi CFSE can be tricky to use. Some cells may indeed take up more then others. We would recommend warming buffers and media to 37oC before using. How many times have you done this and do you have a negative that you can show.?
Hi CFSE can be tricky to use. Some cells may indeed take up more then others. We would recommend warming buffers and media to 37oC before using. How many times have you done this and do you have a negative that you can show.?
As doctor Dunk said, different cell types bind differentially CFSE, then the other cluster represents monocytes, which will not proliferate. Therefore, if the aim is to assess proliferation, the resulting population must be analyzed at the end of the culture and compared against the stimulus. It should be noted that the control will be different from zero day.
I make my CFSE protocol refer to“jove”，and some companies. I incubate the CFSE with cells 15mins 37℃，and stop it by add medium, room temp, then wash.
Because the blood is from myself (it is hard to get volunteers here) ，I can not do it continually.
the first is day zero, the second is PHA neg 7day, the third is PHA pos 7day.
Hi
what you are assuming is correct the cluster which as increased size than your lympho is monocytes. If u want to separate monocytes and lymphocytes I feel you can use the density gradient method or Rosette sep (http://www.stemcell.com/en/Products/Popular-Product-Lines/RosetteSep.aspx).
Then coming to your CFSE stain are you getting different fluorescence intensity for two clusters?
Before staining the cells with CFSE, check whether you have any clumps and after suspending your cells in PBS add CFSE and vortex it immediately.
Incubate cells with CFSE for 5min at 37C that is enough, then add 10-15ml of medium containing FBS and incubate it for 15min (you can also repeat the wash).
you can titrate CFSE according to your cell of interest, I normally use 2-3microM concentration.
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