I have gated unstained SW1116 cells and the majority of the population is within the lower left quadrant. However, there is some streaking that occurs in the upper right quadrant. Could anyone please provide an explanation as to why this streaking occurs? Thanking you in advance
That occur in cell lines with high autofluorescence or doublets. You can exclude doublets by using a FSC-A - FSC-W plot. But in your case i think its the high autofluorescence of your cells.
Hi Shann. Thanks for your response. I did do compensation using single stained tubes. However, the image you see is of unstained and not stained cells.
@Lukas, thank you too for you response. I plotted FSC-A vs FSC-H, gated the singlets population and then ran my unstained and stained samples (FSC-A vs FSC-H). I originally thought it was autofluorescence but I had my doubts. I'm glad you also think it might be autofluorescence, I'm going to go with you on this one. Thanks a mill!
We had the same "trouble" with autofluorescence when we differentiated monocytes to macrophages.
Hi Brenda,
I've also seen this when I have a lot of necrotic cells. Checking with 7AAd and gating out the necrotic cells might help. If they're necrotic, they will stain non-specifically with just about everything.
Fluorescence intensity measurements in flow cytometry are to be appreciated in "arbitrary units", and all distributions we measure have "outliers". Even though you might consider the fluorescence intensity distribution of these unlabeled cells perhaps relatively "large", that doesn't preclude from a correct interpretation of labeled samples. For instance, cells in labeled samples that display orthogonally off the diagonal are single positive, double positive cells will probably increase the percentage of cells in the upper-right quadrant. Rectangular gating is not necessarily the way to go, though …
HTH
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