Recent replies to "CFSE staining"

Reply from M. RAHUMAN SHERIFF

M. RAHUMAN SHERIFF — Thu, 20 Mar 2008 11:06:41 -0000

Hi Aziz,
Hope u got expertise in using CFSE.
I would like to use it to stain HSCs.
Can you suggest me a good protocol for that.

thank you

Reply from Katharine Barnes

Katharine Barnes — Fri, 21 Sep 2007 08:16:43 -0000

Hi Aziz,
You might like to make the most of the fact that for the next month volume 2 of our Nature Protocols content is open access.

Here are a couple of protocols about assaying lymphocyte proliferation using CFSE and analysing by FACS:

Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester

Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data

I hope they are useful. Thanks for being such an active member of our forum.

Katharine

Reply from Aziz Chentoufi

Aziz Chentoufi — Mon, 30 Jul 2007 23:26:46 -0000

Thank u Hilary. Your protocol is quite similar to the one suggested by the manufacturer. But, in many papers they used very low concentration like 1- 2 micM for 10 min at RT. if I use PE-CyAb to stain my cells, it should be easy for compensation?
Thank u for sharing your expertise
Aziz

Reply from Hilary Warren

Hilary Warren — Mon, 23 Jul 2007 06:28:49 -0000

Protocol for labelling PBMC with CFSE

PBMC, either freshly isolated or thawed from liquid nitrogen stored samples are resuspended in PBS containing 5% FCS at concentrations from 0.5×106/ml to 10×106/ml. CFSE is diluted from a stock solution of 5mM (stored -20oC) just before use. Prepare a 1/500 dilution (10 micromolar) and add in equal volume to the PBMC suspension. If the volume is large do the addition whilst vortexing. If the volume is small add the required volume to a ‘non-wetted’ part of the plastic tube, then cap the tube and mix the two solutions rapidly. After 5 minutes add 10 volumes of PBS/5% FCS and centrifuge.

The optimum concentration of CFSE needs to be determined for each batch of CFSE. If the concentration is too high the cells will not divide because of toxicity.

Note that CFSE staining of lymphocytes cannot be measured directly after labelling because of the extremely high fluorescence. The majority of CFSE initially taken up by the cells is not stably incorporated and is lost within the first few days.
Note also that uniform labelling with CFSE is essential to obtain clearly defined peaks following division. That is, the coefficient of variation (CV) of the CFSE-labeled control unstimulated lymphocytes must be small. This is in part determined by the technique for mixing CFSE with the cells, detailed above, and by the uniformity in cell volume of the population.

Normally CFSE analysis on the FACS would be on day 4 after stimulation of PBMC with mitogens.

The exact settings on the flow cytometer will depend on the particular instrument being used. Have the FSC/SSC settings to clearly see the lymphocyte gate, and to have the voltages set so that the background of non-CFSE labelled cells is in the first decade of log. If the cells are to be stained with other mAb, it is important to have CFSE cultures without mAb as well as unlabelled cultures with mAb to be able to set the correct compensations especially between FL-1 (CFSE) and PE labelled mAb (FL-2)