Hi Vox, How long are you waiting between PBMC collection and cell staining? If you are waiting a few hours, the monocytes are probably stuck to your tube. For me, the quicker I stain my sample and then fix the cells, the better monocyte recovery I get during acquisition.

I agree with Nicole Acuff, incubation time should be relatively short (20 mn is largely enough in this case). In addition, I'm under the impression that there is a copious amount of potential dead cells (FSC-low on the left in your gate) when looking at your graphs, so it would be worth trying to optimize your thawing protocol. However, the reason why the addition of anti-CD14 and anti-CD16 antibodies would affect your monocytes is somewhat puzzling....that would help if you gave more details on your experimental procedure.

It is hard to troubleshoot based on the FSC-SSC profiles. I have worked with human PBMCs and the staining protocol is identical regardless of which cell subset you are looking at. I never had issues finding the populations. Like Julien Verdier mentioned, your thawing protocol maybe needs optimization. Monocytes constitute less than 10% and any cell loss might be more obvious in small populations. The other possibility is that your compensations settings might be off. I am leaning towards the latter as you see the problem once you have 3 colors in the tube, not one. It would be help to see your CD3/CD14/CD16 profiles.

Hi Vox,

Have you gated out your dead cells prior to taking this picture of your stained cells? If your monocytes are present prior to gating on live cells, you may be "accidentally" gating out your live monocyte population. This is because monocytes have a stronger background fluorescence intensity when staining with live/dead stains, so they may appear like dead cells even if they are not. Something to consider :)

Additional details would be helpful here, like what protocol are you using to thaw and label your cells? And Scott's question is important too; are you gating dead cells out before the attached images?

It's possible that your frozen cells are dying during antibody and that's why you have monocytes in your unstained sample.

I once ran into a similar situation doing intracellular cd68 labeling by flow which resulted in complete cell lysis (disappearance). I never could figure out what was causing it.

Hello Vox, We found that recovery and viability of thawed human PBMC was dependent on controlled freezing - 1˚C/minute before transfer to liquid nitrogen storage. Then rapid thawing and transfer to appropriate culture media, if required for subsequent culture. Destructive ice crystal are more likely to form in larger cells, in poor freezing media(old DMSO) or rapidly frozen samples. We deplete monocyte/macrophage populations from blood or dissociated tissue, by incubation of single cell suspensions on polystyrene plates, for 30-120minutes. Polystyrene is highly charged and the myeloid cells adhere VERY strongly and allows subsequent isolation of these cells.

If you wish to mitigate myeloid cell loss from your sample. handle your cellular material in polypropylene tubes, as long as you can. I also add BSA V(0.1%) and sodium azide(0.05%) to the basic staining/washing solution, to reduce aggregation and/or inhibit cell surface adherence, while staining for flow acquisition.

Best of luck

Thank you all very much for your answers!

Compensations settings:

Correct me please if I am wrong, as far as I know, dragging the visable spillover with compensation matrix wouldn't affect the very original FSC-SSC plots without any gating or touching (which is the case that I posted) right?

Therefore I am thinking is there any special procedure that I need to be aware of (like fix but not perm the cell before/after surface staining) as Nicole Acuff mentioned.

As I have mentioned I did think about monocytes are probably stuck to the tube.

I still wonder that how would it be possible as I did both staining at the same time with the same protocol...If they stuck to the tube/dead after thawing, wouldn't I be losing them for all the tubes...

Thanks again for all your answers and please let me know if I miss anything or can try anything to make them back to life again!

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Hi Vox, Some more questions: Which clones are you using? Have you tried a tube without CD16 staining? And what is the time between when staining is complete and acquisition occurs?

You've got an honest mystery here.

I suspect that adhesion isn't your source of loss but my suspicions are usually wrong (heh). Though I would always recommend using polypropylene tubes (cloudy plastic), serum, and sodium azide as previously recommended.

As also suggested, I would try unstained vs cd14 only vs cd16 only vs cd14+cd16. While trying this, also as some optimization to your procedure (thawing, bsa, sa, pp tubes).

Fixing your cells will likely prevent your antibodies from binding so that is probably not a good approach.

Compensation won't effect fsc ssc - unless you are compensating these parameters. If you are, then don't.

Hope this works out. Seems frustrating.

Hi guys I have figured it out!

It was the TUBE...

You are right Daniel McKim polypropylene tubes works perfectly this time.

If this really is the case then they will only stick to the tube after being labeled/activated/touched, but I still wonder why they will.

Thank you all for the excellent answers and suggestions!