My samples are a mix of cancer cells and lymphocytes. I usually do doublets discrimination when performing data analysis but I would like to know if a heterogeneous doublet formed by a cancer cell of 18 um of diameter together with a lymphocyte (7 um) can still be discriminated in a plot FSC-H vs FSC-A? I don't think so since after doublet discrimination some of my cancer cells (based on size) result positive for CD45 (pan-leukocyte marker). What is your opinion about it?
Hello Giulio,
Do you plot FSC-A vs SSC-A before discriminating doublets? I take it that you do but you were unable to distinguish them that way! If so, do you have access to homogeneous samples that you can use as controls? In the event that you do, you could always use them to establish the gates corresponding to each of your populations.
Hope it helped,
João Nina
Hi Giulio,
I think you may also do doublet discrimination based on the DNA content.
If the cells are fused due to cancerous nature, polyploidy or simply stuck together, the DNA content associated with the event will give you additional parameter to distinguish singlets from doublets.
DNA content can be evaluated using any DNA nuclear dyes such as PI.
Best
João Miguel Nina I first do FSC-A vs FSC-H. The problem is that already in an homogeneous population the shift of the doublet events in the plot is minimal when cells have the same dimensions. If cells have very different dimensions I think it won't be possible because the difference in diameter and the time that the event need to pass the laser (how they asses Area) it won't change that much in between a singlet and a doublet. Thanks anyway.
Hi again Giulio Giustarini ,
I asked you whether you started by plotting FSC-A vs SSC-A simply because that will allow you to distinguish cells according to size and complexity/granules. I don't think lymphocytes and cancer cells share those features, do they? Therefore you would be able to gate your populations using the first plot (FSC-A vs SSC-A) and then you distinguish singlets from doublets within the gates (FSC-A vs FSC-H).
I was suggesting you used homogeneous samples as a control to realize which population would be which, when you plotted FSC-A vs SSC-A. However, if your cells differ that much in size within their own...
Best regards,
João Nina
Giulio Giustarini ,
I believe, it's feasible. You need to get some parameter of controls at the beginning. 1st control - Normal cells , 2nd control - Cancerous cells, 3rd - Heterogeneous cell population . Now, start analyzing them by FSC-A vs FSC-H and you will be able to differentiate them. Except these , you can always use Propidium Iodide to distinguish normal vs cancerous cells.
BR,
Babu
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