Hello
I am optimising cell proliferation assay of human T cell using this dye from Thermofisher:
https://www.thermofisher.com/order/catalog/product/65-0842-85
I have done two groups; one T cells activated with CD3/CD28 Dynabeads and the second group in medium only with no stimulation. I followed the proliferation everyday for 5 days and analyzed the results using Flowjo software. I have attached a copy of the results. The point which I am not sure about is how to interpret these result. What I did was just gating the day 0 staining on the right as the parent cells, and on the left as the divided cells, but is that enough.? I have tried ModfitLT software but I found it is not very helpful with my results as I can't see distinct peaks!
Any suggestions will be appreciated
Abdul
Dear Abdulrahman Bahashwan,
If the red histograms show the data on day 5, your assay might have been at least partially successful. Peaks are not clear but somewhat detectable.
For reference, below is the link to an article doing a similar assay using CFSE.
Article Disparate In Vitro and In Vivo Requirements for IL-2 during ...
In the article, the authors stimulated CD8 T cells for 0-18 hours with anti-CD3/CD28, transferred the cells to another plate and cultured for 4 days (including the hours of stimulation).
Fig.1 suggests that the culture for 4 days is appropriate to measure T-cell proliferation in vitro. 5 days may be OK as well.
If you feel that cells were dividing too quickly in your exps, then you could optimize the stimuli (ab titers and/or duration) to better separate the peaks.
By the way, I would suggest comparing between unstimulated T cells and stimulated T cells at day 5, rather than comparing among unstimulated or stimulated samples at different days.
So, congratulations for your assay that have been basically successful. Following optimization will make it perfect for your needs.
Following link may be useful
https://docs.abcam.com/pdf/primary-antibodies/the-cell-proliferation-guide.pdf
Here are a few things I have done to help. Be sure to have a viability dye included when you analyze proliferation. If you want distinct peaks you can try something called the sort reduction method, essentially you stain with your CellTrace and sort a portion of the histogram with a small gate (CV around 20), your parental peak is pretty wide so any subsequent peak will be lost in the large CV. I would add an unstained histogram to make sure that the background does not overlap with your results. My guess is that your background from unstained cells is not below the axis as your gate suggests. You want to avoid interpreting results that overlap with the background.
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