CFSE labeling of human PBMC - proliferation assay (Jun/20/2003 )
Hi
I have labeled cells with 3.16 uM CFSE for 5 minutes, washed, and setup cells for culture (stimulated cultures using 1 ug/ml of SEB). After analysis by FACS, I had expected to see several peaks on the histogram of CFSE after 3 days of culture indicating proliferation. However, there is only a single peak ?
Why dont I get several nice peaks showing each cell division ?
If anyone has got experience using CFSE on human PBMC please comment.
Big thanks,
Tom
-erenskjold-
Hi,
This was actually quite old post. Are you still there?
I have the same problem, and I am interesting how have you solved it.
Sincerely,
Lev
QUOTE (erenskjold @ Jun 20 2003, 04:01 AM)
Hi
I have labeled cells with 3.16 uM CFSE for 5 minutes, washed, and setup cells for culture (stimulated cultures using 1 ug/ml of SEB). After analysis by FACS, I had expected to see several peaks on the histogram of CFSE after 3 days of culture indicating proliferation. However, there is only a single peak ?
Why dont I get several nice peaks showing each cell division ?
If anyone has got experience using CFSE on human PBMC please comment.
Big thanks,
Tom
I have labeled cells with 3.16 uM CFSE for 5 minutes, washed, and setup cells for culture (stimulated cultures using 1 ug/ml of SEB). After analysis by FACS, I had expected to see several peaks on the histogram of CFSE after 3 days of culture indicating proliferation. However, there is only a single peak ?
Why dont I get several nice peaks showing each cell division ?
If anyone has got experience using CFSE on human PBMC please comment.
Big thanks,
Tom
-levli-
QUOTE (rashmi @ May 3 2005, 03:26 AM)
hi
i say that i was succesfull in getting those peaks for the cell division using CFSE labelling. if you can give me your mail id i will post my protocol.
following are the conc
4uM of CFSE
5ug/ml of SEB for PBMC
the only problem that i faced is the compensation for the CFSE in FACS calibur.my id
rashmi.nagaraj@gsf.de
cheers
rashmi
i say that i was succesfull in getting those peaks for the cell division using CFSE labelling. if you can give me your mail id i will post my protocol.
following are the conc
4uM of CFSE
5ug/ml of SEB for PBMC
the only problem that i faced is the compensation for the CFSE in FACS calibur.my id
rashmi.nagaraj@gsf.de
cheers
rashmi
Hello, Rashmi
I see you quite experienced in CFSE proliferation assay of human blood.
I am doing an CFSE MLR in mouse system and also do not see any peaks. I will have to do CFSE MLR with human samples as well. The altimate goal is to sort out dividing population based on CFSE dilution. Iam expecting a lot of problems because the dividing population is very small.
If you are familiar with this issue, please give suggestions or may reffer me to somebody else. Also, I would really appreciate if could send me a protocol for CFSE labeling Human PBMC.
Thank you very much.
Hermine.
-Hermine-