I have stimulated whole blood with LPS and the cells on the top gate (very high SSC) show up regulation of the intracellular protein i am investigating. The SSC looks unusual but as you can see from the image, most of these cells have normal FSC and express monocytic markers (classical and intermediate subsets). Any suggestions?
Further to Yumeng's comment, I'd reduce/adjust the flow cytometer voltage next time. This high SSC to me is off the scale. Also monocytes normally are low/medium for SSC, so, you shouldn't see monocytes there.
Hope that helps.
I don't know much about flow cytometry of blood cells, but in order to know if gate your data with only live cells I have used a live cell dye, such as IP or TO-PRO-3. These dyes can only get inside cells if their cell membrane is damaged, ie, dead cells.
Hi Oscar
I have used a fixable viability staining which shows that at around 70% of those cells are viable, my concern is that maybe it's non specific binding or not cells at all (debris or others).
Hi, have you checked the CD45+ cells that had the SSC above 100K? I am not 100% convinced that it is a good idea to set your gate on the cells that out of your upper range since the cell counts are too low to give you trustworthy results. I would gate CD45---live/dead---Singlets---CD14 and then plot HLA-DR against CD16. This way it helps you to get rid of B cells, which express also HLA-DR and CD16. Good luck!
Further to Yumeng's comment, I'd reduce/adjust the flow cytometer voltage next time. This high SSC to me is off the scale. Also monocytes normally are low/medium for SSC, so, you shouldn't see monocytes there.
Hope that helps.
I agree with Yumeng, Mahmoud and Kevin re the SSC signal. In all probability these cells are aggregates. Your single cell gate isn't showing that as you really don't have a single cell population present. Yumeng's gating strategy is a nice simple strategy. You might also want to backgate your 14+16+DR+ population for display on a FSC/SSC plot to see if these cells show up in the expected monocyte region.
Hi, Nunzio!
You used whole blood, right?
I think you don’t have to adjust the settings of you cytometer, because you have all the cells in the right position. Looking at the first image (SSC vs CD45) we can nicely identify neutrophils lymphocytes and monocytes. Though I think you should lower your SSC threshold, because you have cut off part of the neutrophils and lymphocytes.
Concerning the cells in the gate above, I would exclude them.
After LPS activation and incubation at 37.ºC, monocytes get a little bit stranger, but not that much... Cell debris and apoptotic cells can bind to mAb in a non-specific way; and debris also bind to surface receptors on viable cell. Moreover, in the CD14 vs CD16 dot plot, those gated cells don’t seem right, because you cannot see a continuum from classical to intermediate and to non-classical monocytes.
If you are studying cytokine expression, it is better to have a lower number of monocytes to analyze, but be 100% confident that the events your are analyzing are in fact monocytes.
And I agree with Lynda: looking at FSC vs SCC dotplot will help you a lot.
Best wishes!
I agree with the concern with respect to the SSC signal. Also, your doublet discrimination gating is not ideal for detecting singlets. This workflow from BD:
https://www.bdbiosciences.com/documents/facsdivav6_aria_quickrefguide.pdf
shows proper doublet discrimination gating (FSC-H vs FSC-W and SSC-H vs SSC-W).
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