2 replies to this topic

[Angelie]

Hello,

If someone has used the Flow jo proliferation platform to analyze the CFSE stained and stimulated T cells, I have a question for them. I am not seeing very clearly defined peaks for my CFSE dilution assay as I expected. The peaks are very indistinct and quite mingled together. Can anyone tell why this is happening? Is that something to do with the CFSE staining or washing or the no. of cells used for CFSE staining. Does anyone know what contributes to the clearly defined peaks in the CFSE proliferation data.

Thanks,
Angelie

[Rsm]

You should use non-cycling (resting) cells for staining. If you have a proliferating cell line or activated lymphocytes, those will give you a very broad peak, without clear populations. Label intensity is correlated to cell size, so resting cells = uniformely small, prolferating cells = small, medium and big cells.

rsm

[Denis Baev]

Angelie, on 20 October 2010 - 08:12 AM, said:

Hello,

If someone has used the Flow jo proliferation platform to analyze the CFSE stained and stimulated T cells, I have a question for them. I am not seeing very clearly defined peaks for my CFSE dilution assay as I expected. The peaks are very indistinct and quite mingled together. Can anyone tell why this is happening? Is that something to do with the CFSE staining or washing or the no. of cells used for CFSE staining. Does anyone know what contributes to the clearly defined peaks in the CFSE proliferation data.

Thanks,
Angelie

Sinchronyze you cells. You will never see good CFSE pikes if you stained freshly isolated PBMCs for ex.