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CFSE staining protocol - (Jun/27/2009 )

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What kind of results did you get?

-miBunny-

miBunny on Aug 11 2009, 03:50 AM said:

What kind of results did you get?


I think that I didn't stain the cells, because I couldn't find any fluorescence in FL1. What I tried it was to add 10 ml of 5 uM CFSE to a petri dish with PC-3 cells attached, leave it at 37 degrees and then replaced it with RPMI 10% FCS, leave 30 min and the replace with other RPMI 10% and leave for 3 days

-canotto-

I think that I didn't stain the cells, because I couldn't find any fluorescence in FL1. What I tried it was to add 10 ml of 5 uM CFSE to a petri dish with PC-3 cells attached, leave it at 37 degrees and then replaced it with RPMI 10% FCS, leave 30 min and the replace with other RPMI 10% and leave for 3 days


That should work. It is definitely feasible and the way you did it should have worked. Were you comparing the labeled cells to unlabeled cells? Cultured cells often have a lot of autofluorescence but with 5 uM CFSE you should see a big difference between stained and unstained. If you collected the PC3 cells using trypsin it is also possible you trypsinized the label right off the cells. You might want to try labeling in the dish like you did and in parallel label in suspension. Don't wait 3 days, just go ahead and analyze using unlabeled cells for a baseline control.

Good luck.
Attached File

-Astarte Biologics-

Hi!

I used CFSE before to stain T cells and DCs, but now I want to track Mycobacteria (BCG) with it for >14days in vivo.
The Mycobacteria have quite a thick cell wall and are sticky guys. I found a paper in which they label M. tuberculosis with CFSE and they state they use the normal PBS with 0.05% Tween 80 and stain for 60min at 37C. What they don't say, unfortunately, is what concentration of CFSE they use. I guess you would need more than for DC or T cells because of the thick cell wall? And they also stain a lot longer than we do for DC. For DC we use 1uM CFSE for 10 min at RT and then wash them twice.
Has anyone here tried staining mycobacteria with CFSE? Any suggestions for the concentration? I would like to be able to still see them after 2-3 weeks (and I don't know how fast the dye is fading in non-dividing cells) but I don't want to kill my BCG off with overloading them with CFSE. Unfortunately, trying different concentrations and looking at CFU will take ages as they take 3 weeks to grow to colonies on the plates...
Can anyone help me?

Thanks,
Binchen

-Binchen-

do you have any plot of FSC/SSC and FSC/CFSE on ConA stimulated PBMCs ?

Since I don't have an anti-CD3 antibody, I don't understand exactly where to put my lymphocytes gate ( of course, stimulated lymphocytes are bigger than nonstimulated, but I don't understand how much. Do they overlay with macrophages?)

Thanks

-canotto-
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