CFSE does not show distinct peaks - (Jun/16/2011 )

Hey guys,

I am using CFSE staining for freshly isolated hematopoietic stem cells (HSC) to track cell divisions/proliferation. The problem is, after 7 days of culture I cannot see distinct peaks for the genereations.
You can compare the result here:

http://dl.dropbox.co...%207%20HSC.pptx

Has anybody experience with CFSE?

Thanks
Cerise

Cerise on Thu Jun 16 08:54:16 2011 said:

Your link doesn't work....
CFSE is always troublesome for primary cells, because initial staining intensity depends on cell size and metabolic activity. Dormant HSC are small and most likely resting: weak signal. Activated HSC are big and metabolically active: strong signal. This results in broad distribution of the initial signal. Better use BrdU or (Tet) H2B-GFP. But that's also a bit tricky...

sorry for the dead link, I attached a picture of the CFSE peaks now.
okay, so do you have any idea how to improve the staining for my HSC?

So far I do it as follows:
- centrifuge HSC and aspirate supernatant
- 750 µl of PBS containing 0.1 % FCS and 250 µl of 7.5 M CFSE (final conc. 2.5 M) solution are added to the cell pellet and incubated for 10 min at 37 °C
- the staining is quenched by the addition of 5 ml of ice-cold PBS containing 10 % FCS
- cells are incubated on ice for 5 min and centrifuged down
- cells are washed by resuspending in fresh media and incubated as usual at 37 °C and 5 % CO2

I saw in other publications that it should work this way...

I also saw it in other publications this way, but couldn't reproduce their result. Is the publication you mention done on HSC (or resting T-cells, what most people use for CFSE)? You can try also DiI, which is somewhat different (but not much better in our hands). Sorry to be of no more help, but at least you now know that you're not alone...

Rsm on Mon Jun 20 14:06:37 2011 said:

hehe, thanks. This helps a lot. At least for my motivation. The picture is actually copied from the manufacturers instructions. But there is some publication where it worked just as good with HSC (Pubmed Reference: 21437259). Check it out. The protocol is exactly the same.
Everytime I tried to find an answer to this problem people recommend me to use another method. What is DiI?

-Cerise-

CFSE stains and reading is definitely tricky. The 'optimal' separation of CFSE pics at the end of your experiment depends on a numerous parameters including: staining protocol, cell type you are staining, numbers of days of your assay, antibody panel used in combination with CFSE, compensations and cytometer performance. Although I never did HSC but only T cell CFSE assay, you protocol looks good but the concentration of CFSE you are using looks very very very high ... I use concentrations in the µM or even nM range and this can definitely affect your result at the end. To my experience, the lowest dose you use to stain, the better it will look at the end (plus it will be a lot less toxic). Try to titrate it down if you can.

-alwaysbreizh-

Cerise, your CFSE staining looks fine. Actually it looks pretty darn good. You'll never get separate peaks and analysis of such data requires modeling such as what is available in software like ModFit (verity software house) and FlowJo (Treestar).

-RynDggn-