Need protocol: CFSE Staining/ primary cells - (Aug/11/2006 )

Hi there,

I would like to perform proliferation assays with primary hepatocytes by CFSE staining and analysis performed by flow cytometry. I tried myself different CFSE concentrations but I always obtain broad hills in my histogram instead of sharp peaks. So my question: Could anyone provide me with an established protocol to enable my to calculate the proliferative index of my primary hepatocytes.

Thanks in advance and best regards,

Bob

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Hi Bob,

As far as my experience goes with CFSE it is really difficult to get sharp hills when cells are not sychronized.

To track cells in vivo I labeled mouse lymph node T cells with CFSE before passive transfer into syngeneic recipients. Then, I analyzed T cell homing of selected T cell subpopulations by FACS.
Although the peaks were somewhat broad I could distinguish at least two peaks (given the short time between passive transfer and cell recovery).

The explanation I find for the broad peak is that CFSE does not synchronize cells as BUDR pulse labeling does. Therefore, it is difficult to obtain sharp hills unless some kind of synchronization exists (i.e., chemical or physical methods).

I encourage you to use FITC-anti-BUDR/PI after BUDR pulse labeling if your interest is to establish cell cycle progression (i.e., Td, Ts) and not only the number of doublings.

For BUDR labeling I used the protocol developed by Dolbeare (Proc Natl Acad Sci U S A. 1983 Sep;80(18):5573-7). You can get the full reference from PUBMED.

Anyway, here is the protocol I use for CFSE labeling:

1) Cells are resuspended to 1x107/ml in 1X phosphate-buffered saline, pH 7.2 (PBS), containing 5 µM CFSE (Molecular Probes, made up from a 5 mM stock in DMSO).

2) After 5 min at 37 ºC, labeling is quenched by the addition of an equal volume of fetal bovine serum, and the cells are washed twice with 1X PBS, counted and resuspended to the desired concentration to plate or transfer in vivo.

Hope this helps. Best regards,

Gerardo

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Hi Gerardo,

many thanks for your supportive reply. Maybe I will simply do a channel gate sorting shortly after cfse staining prior to seeding the cells to sharpen the resolution of my histogramm meaning that I want to select/ define synchronized cells by roughly the same initial fluorescence intensity. Parallel I will try to synchronize my cells chemically hoping that it leave them somehow unaffected for my subsequent planned stimulation experiments.

Best regards,

Bob

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