Due to the dead cells in your sample a DNase treatment is advised to avoid clumping. But I don't expect any further problems with surface staining and flow cytometry.

We do not expect the cells to remain viable that long. Anyways, you can fix the cells using 2-4% paraformaldehyde (PFA) for 30 minutes, wash out the PFA and directly continue with the staining for surface markers.

You can try that - First, wash your cells (eliminate the DMSO). Then, use your cells marker antibodies needed for your experiment and stain your death cells with a viability marker (Propidium Iodide and 7-AAD can be used to stain dead cells). With that you will be able to dissociate your cells of interest.

I think it all depends on the percentages of dead cells in your PBMC population.  You can take a sample and do a quick tryptan blue stain to first estimate ifa major part of your population are dead or alive.  In the latter case, i don't think you should have any problems with carrying on your flow.  If not (i.e if the majority of your cells are dead) and you could estimate that you have enough live cells left to do your flow, then you can get rid of the dead cells by simply spinning at very low RPM (300), ideally you shoud get rid of the dead cells that way (as you will be left mostly with live cells in your pellet while dead cells can be discarded with the medium- dead cells are generally lighter than live cells,  that is why they ideally should stay in the surface)...hope you got my point.

I do not think the flow cytometry results would be reliable, so it depends on what you intend to do with the results. 

Some MNC sub-types will be more affected by the sub-optimal storage conditions than others, so your viable cell population will be biased towards the resilent sub-types. Also there will likely be patient-to-patient variation in the level of resilience and this will be further affected by their medical condition(s), medications, etc, etc.

If you want to investigate frozen cells, I would suggest you for the future to freeze and store them at least at -80°C (short time), better in liquid nitrogen or in at least an -150°C freezer for long term storage.

I am not sure if you can obtain your desired results as freezing at only -20°C is not sufficient and most probably a lot of cells will be dead...

Hi,

After thawing down, lot of cells are going to be death (mostly B cells), but do your staining and analyze the live cells gate (PI staining or by FSC/SSC). I agree with Daniela Bruennert that is better to store frozen cells at -80 or liquid nitrogen.

Good luck!