Currently, we are trying to measure proliferation speed in fibroblasts from healthy donors and patients. We are trying this with Celltrace violet. We tried different concentrations, 2 uM, 5 uM and 20 uM for 200.000 - 500.000 cells. We incubate cells for 20 min at 37 degrees, and measure after 3/4 days. However, during FACS cytometry we do not observe the nice "peaks''. What am I doing wrong?
When it is flow-cytometry many things may have gone wrong. But you must give more details to figure out the problem. When you say " We incubate cells for 20 min at 37 degrees" it is the labeling step? If so, are the concentrations there concentrations of the dyes? Why 3/4 days? Do you see cells with forward/side scatter in flow cytometer? Do you get any fluorescent signal?
Thank you for your reply!
The labeling step is 20min at 37 degrees. Then 1 wash step with FBS/medium. We measure CTV after 3/4 days because we want to see 3/4 cell divisions, and our fibroblasts divide aproximately once a day. We get a strong fluorescent signal, but no peaks, just a pink mountain so to speak. We get a forward and side scatter, and are able to gate for dead cells (with PI), doublets and debris.
If your cells are visible in fwd/side scatter screen, and you can place gates to get rid of the debris/dead cells, and have a fluorescent signal, I would suspect the flow cytometer settings instead of the protocol. Theoretically, fluorescent signal should have 4 peaks, one for each generation. Last peak (highest fluorescent signal) should have lower height (the number of cells from that generation is low) and the first peak (last generation/lowest signal) should have a higher peak (more cells). If the differentiation is not clear it may look like a "pink mountain". The fluorescent scale should be logarithmic if not, it may be harder to notice. You may also need to check voltage parameters of the detectors to get an optimal result.
Hi.
I have the same problem trying to measure proliferation in fibroblast-like synoviocytes from rheumatoid arhritis joints. I get a signal but no separation between the generations. Did you find a way to solve the issue?
Hi, I am having the same issue with melanoma cell lines any solutions?
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