Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester
This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.
Comments
A question relating to this protocol was asked on our Discussion Forum. It can be found here: http://network.nature.com/groups/natureprotocols/forum/topics/5102
I have duplicated the exchange here:
Biplab Bose: We are doing cell proliferation assay using CFSE statining, followed by FACS analysis as per the protocol published in Nature Protocols.
We are measuring the fluorescence intensity of CFSE stained cells in FL1 channel of the FACS. But am bit confused whether to collect FL1-A data or FL1-H data . As I understand one should collect FL1-A data to avoid the effect of change in cell size during cell cycle.
However, I do see many publications where they have collected FL1-H data and have shown that as a histogram. Most of the publications simply put it as “CFSE intensity”, with out saying FL1-A or FL1-H.
It will be nice if you can give some advice on this.
Ben Quah: FL1-A measures the area of the pulse signal, whereas FL1-H measures the height of the pulse signal. In addition, some flow cytometers can measure the width of the pulse signal (eg FL1-W). These measures can apply to all parameters. As long as you exclude doublets from your analysis, FL1-H and FL1-A should be fine to measure your CFSE intensity.
Posted by: Bronwen Dekker | October 25, 2009 10:19 PM