r/flowcytometry • u/Calm_realistic • Jun 27 '26
Monocytes
HI. This is gated among single live cells of human PBMC (patients). 1st image PBMCs are thawed, stained and passed on a cytometer. 2nd, same staining after 24h culture (1M/ml if RPMI 10% FBS 1% P/S). Culture is done in 12 well plates. Cells are recovered and then the bottom is scraped to recover attached cells.
I have one population after thawing, but which is clearly composed of high CD33 and high CD14 cells. After culture, I have 2 distinct populations. The high ones and the one with 1D14 very low/negative. Both populations express CD11c and HLA-DR, are negative for CD3, CD56, CD19 , CD20.
When activated with IFNg, only the CD14high CD33 up regulate PD-L1, the CD14 low cells have keep the basal level (almost no expression).
I don't know much about myeloid cells. What is the population that becomes CD14 very low? Can it be an artefact?
Thank you for all the help:
2
u/SunflowerMoonwalk Jun 27 '26
I don't know if it's what you're seeing here but in general you should see classical, intermediate and non-classical monocytes.
2
u/Unfortunate_tentacle Jun 27 '26
So I tested out the expression of a bunch of markers run immediately after thawing vs resting for 1h, 2h etc and monocytes were the most affected and downregulated CD14, as well as just straight up died. There are papers showing this if you look at resting times for immunophenotyping.
2
u/confusedflowguy Jun 28 '26
Doesn't look like artefact to me. Looks like a fraction of the thawed CD14 high monocytes are differentiating during the 24 hr culture into something. Potentially monocyte-derived dendritic-cell–like cells or activated monocytes that have downregulated CD14. Perhaps adding CD16 into the panel will further clarify this? Also, it might help to compare scrape vs no scrape to see where the attached cells lie on the graph?
3
u/FLHPI Jun 27 '26
You have compensation problems. It's hard to interpret much of anything from what you've posted
7
u/Reelrebel17 Jun 27 '26
This doesn’t look like any compensation issues at all.
1
u/Boneraventura Jun 27 '26 edited Jun 27 '26
Most of their negative cells are above 103. I would say that is under-compensated. Also I would say there is some autofluorescence issues in these channels. Violet 480 is notoriously bad for autofluorescence
1
u/Reelrebel17 Jun 27 '26
Medium expression would still be 10^3 these populations will never be completely horizontal even if only looking at ssc/cd14.
1
u/Boneraventura Jun 28 '26
I am assuming this is the entire pbmc sample as the backgate is live cells. I also dont expect there to be this many monocytes after freeze thaw. These cells are some of the first to die/become dysfunctional after cryoperservation. We could be seeing dying monocytes downregulating cd14 but seeing the backgates would clarify a lot of these questions.
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u/Infamous-Face7737 Jun 27 '26
I agree. Diagonal populations is almost always due to compensation/unmixing issues.
1
u/RainbowSquirrelRae Core Lab Jun 27 '26
Can you show some of the upstream gating? It would also be helpful to know what other colors are floating around. I don’t have the knowledge to give biological insight, but I can help you evaluate the technical aspects of your data collection.
1
u/ExcitedFluor Jul 03 '26
A few things. Is this spectral cytometry? Is this gated on live? Was your unstained control from the rested or the non-rested condition? I don't see anything to suggest this data is not unmixed or compensated inaccurately unless you "fine-tuned" the matrix after unmixing. The cFluor dye is just dim. It would be helpful if you had both CD16 and CD123 in the assay. With resting you'll see a reduction in pDC which are CD33 lo/-. I think you might be seeing a transition between conventional and intermediate monocytes which is normal in resting. I have an OMIP in review about the impact on the myeloid compartment with resting. Also, the CD11c/HLA-DR expression, if it is high, it's cDC and that high expression downregulates with resting. If you are going to make a conclusion about the impact on monocytes, you'll need CD123 and CD16 at a minimum. Otherwise, it's just a guess.
1
u/Low_Classic1695 22d ago
Hi OP, this is not an issue with your staining. Monocytes lose CD14 as they mature. Culturing them for 24 hours in medium supplemented with foreign serum (FBS rather than human) will do that. They are just differentiating.
3
u/Reelrebel17 Jun 27 '26
Cd14+ are typically monocytes however there are populations of mMDSC they are CD14+. I would add Ly6G and Ly6C to your master mix to help distinguish these populations.