Hi everyone,
I'm hoping to start working on some dye dilution proliferation assays using flow, I'll be using the life technologies cell trace violet kit. I was wondering if anyone has any experience using this kit?
I'm trying to track cell divisions in bone marrow cells as they differentiate with MCSF to become macrophages. I'm not sure about protocols and I've found several online but does anyone have any first hand experience doing these assays and would be able to give me some tips?
Thanks.
Samatha,
I have never used the violet kit before but I have stained cells with CFSE. I have adoptively transferred the cells into a mouse and then harvested cells back out. I then stained my cell subset of interest (with PE or APC since CFSE is green). I then run the cells on the flow machine. Setting the FL-1 channel to linear scale, you can see the large peak on the right (no proliferation) and than the intensity of the CFSE is diluted by 1/2 for every cellular division. So the peak just to the left of "no proliferation" is 1 division. The next is 2 divisions, etc. You can then analyze each peak to see what % of cells have divided, not divided, or divided a certain number of times.
Best of luck!
Hi Samantha,
We are using CFSE, PKH and eFluor670 proliferation dye (eBioscience) for in vitro proliferation assays. There are some things you have to consider: Once you have stained the cells with proliferation dye you have to measure time point 0 on the FACS. For freshly isolated and stained cells you should get a sharp peak in your FACS histogram. Now you have to adjust this peak to a "defined intensity" by adjusting the PMT voltage. These settings should be saved because they define the starting point of your kinetics. Be sure to use the same settings for all subsequent measurements of the kinetics. There are some things you have to keep in mind as well: 1) Some dyes (CFSE etc.) are actively and/or passively exported out of the cell (which has nothing to do with proliferation!!). What you will get then is a deacrease of fluorescence intensities which is not related to cell division. 2) Some dyes might be toxic at certain concentrations (you should test that for the cells you want stain).
Best wishes and good luck
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