PROT1 vs PFA for sample storage & shipping

[javells]

Dear All,

A collaborator will be performing CyTOF surface staining on treated samples and then shipping them to us after cisplatin labeling and fixation. I have a few questions regarding the fixation process:

What is the optimal fixation solution for storing fixed cells at -80°C and shipping them? PROT1 or PFA?
How long can PFA-fixed cells be stored at -80°C without any proteomic changes? any experience on this ?
Has anyone used PROT1 for isolated cells/PBMCs rather than for whole blood? If so, what is the recommended amount to use? I've reached out to the company but haven't received a reply yet.

Best,
Muharrem

[mleipold]

Hi Muharrem,

Honestly, you need to do testing with your study.

We have generally had good luck with PROT1, but we've only done it in the context of WB studies, not PBMCs. Even in the case of WB, though, we have had some troubles with collection sites not following the fixation and freezing protocol properly, leading to clumped samples or over-fixed RBCs, all of which were unusable had to be tossed. Neither of those issues should affect PBMCs, but still best to try out.

We have not played much with freezing 2% PFA-fixed cells. If you do try that, I recommend that you fix, then freeze in FBS/DMSO: we did some trials for my Multicenter paper where we just froze prestained samples in 2%PFA+PBS, and we started losing a small percentage of B cells and Monocytes.

I have used PROT1 on PBMCs, but just as a small pilot for someone. In that case, I didn't freeze the sample after (that would have been step 2 of the Pilot), so I can't comment directly on recovery out of frozen PROT1 samples.


Finally: we haven't worked with it much ourselves, but Petter Brodin's lab and others have used Cytodelics buffer rather than PROT1. Again, I think they were in the context of WB. I do know that PFA vs Cytodelics vs PROT1 can affect the staining of different markers (especially relative to Live), which is again a reason for you to do your own testing.


Mike

[eganio]

Hi Muharrem,

We have used PROT1 on PBMCs with subsequent freezing at -80C several times successfully. We actually found staining to be improved with PROT1 vs. 1.5% or 2% PFA in PBS as a fixative. We re-suspend PBMCs in PBS at 1-2M cells/mL and use the PROT1 accordingly as if it were whole blood (i.e. 1.4mL PROT1 per 1mL PBMC suspension).

Hope that helps.

-Ed