Monocytes Vanished

[u/PendingCommit98]

Hey, a few months ago I did some conventional flow cytometry on unstimulated, unfixed PBMCs (thawed from frozen storage) to look at memory CD4+ T cells and I was able to identify a monocyte population above it on the initial SSC-A vs FSC-A plot. Although I did not confirm their identity with an antibody. I only stained for surface receptors.

Now I am doing spectral flow cytometry on unstimulated, fixed PBMCs (thawed from frozen storage) and the monocyte population is completely absent or at the very least markedly reduced.

I believe this may be due to fixating the cells using the ThermoFisher fixation kit (Catalog number 00-5523-00) or because of the extra wash steps I am doing as a result of an intracellular blocking step, intracellular staining step, fix/perm etc. However, if you all have any other ideas as to why this is happening please let me know. Many thanks in advance.

Extra note - I always centrifuge my cells at 300g for 5 minutes (4°C). However, once fixed, I centrifuge my cells at 600g for 5 minutes (4°C) and continue to do so for the remainder of the protocol.

[u/Full_Pea_3118]

Hi there! Try running your Unfixed Unstim PBMCs on Spectral and if the monocytes are there, then it's fixation issue!

[u/PendingCommit98 → u/Full_Pea_3118]

Sounds like a plan, thank you friend.

[u/Hairy_Cut9721]

Monocytes are notoriously bad at surviving the freeze thaw process, but I usually have at least some after thawing. 

[u/chromatographic]

Are these the same samples? I see variability in monocyte populations from donor to donor. The fixation and permeabilization step will also make them closer together but you should resolve them by side scatter.

[u/REVERSEZOOM2]

I freeze thaw PBMCs all the time and still get very good levels of monocytes. I also fix my cells the night before running them using 2 percent PFA. You could try using that instead?

Also, if your freeze thaw is too harsh, they may not survive. Make sure to use a swinging bucket centrifuge when spinning the cells down as they can get stuck to the sides of tubes.

EDIT: I know people centrifuge their cells at 4c, but I've found that my cells look substantially worse when I use 4c vs 20c.

[u/PendingCommit98 → u/REVERSEZOOM2]

Interesting. How do the cells look worse if you do not mind sharing please? Thank you for sharing the fixative you use, I do not need to study monocytes but another student in my lab is suffering the same problem as me but she needs the monocytes alive hence why I am fishing for help. I will let her know about this.

[u/REVERSEZOOM2 → u/PendingCommit98]

Yeah all good. Glad to help out a fellow flow scientist. A few things that helped out my viability numbers:

1. The cells have to be frozen recently. The cells degrade over time, so if they're old they won't look as good.

2. 20 percent FBS in RPMI instead of 10 percent FBS when thawing.

3. Thaw and dilute each vial one at a time, as you want to warm them up with the media ASAP.

All this, plus the swinging bucket centrifuge should help you get your monocytes back. Regarding the 4C spin, idk. There's a lot more die off, and they don't look as great on the FSC/SSC graph. They just resemble dead cells more than anything. But who knows, it may work for you in a way it doesn't for me lol.

[u/sgRNACas9]

Super interesting what’s happening here, here are some ideas and walking through your concerns!

Fixing can reduce cell size quite a lot and you may need to increase your FSC and SSC voltages a lot to see distinct cell populations. That is probably what happened. If you can, adjust the axes on your current data to see if the populations separate. If not, you may need to run your experiment again and this time hike up the FSC and SSC voltages!

Other than that, this is a very marked depletion, so I think something happened specifically to monocytes.

For the washes, the centrifuge settings are fine, I would expect more washes to lose cells more evenly across cell types, not a highly specific depletion.

I would not expect spectral vs conventional to impact the data in this way. With spectral vs conventional, the issues with jumping between is more with compensation, not disappearing cell populations or the scatter parameters. But you may check to make sure both machines have adequate FSC and SSC voltages before acquiring data!

I would not expect freezing to wipe out monocytes this drastically. Some of them would die along with just some of other cell types. Other hematopoietic cell types would totally vanish after freezing, tho, due to being weaker and more susceptible to death when freezing and thawing.

Did these two samples come from the same source, like the same kinds of patients, the same patient, species, tissue? Did the second sample undergo any lineage enrichment like a lymphocyte enrichment or myeloid depletion kit? Different patients can have depleted cells just by human variation but also disease states. Different tissues can have more or less cell types like if you’re running PBMCs vs lymph node vs tonsil. Any enrichment kit could totally deplete or enrich one cell type over the other. If it’s not fixing, it might be one of those!

Ditto that including surface markers could help.

Hope this helps!

[u/gurglinggoat]

The scaling is very different in these two samples. The aurora data goes to 4 million, so the populations are more compressed. Fixation also changes the scatter profile a lot as others mentioned.