Fixing/Freezing Metals
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It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style
It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style
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Are metal conjugated Abs stable after fixation and freezing at -80? Does anything happen to metals kept at low temperatures for a long time?
Re: Fixing/Freezing Metals
When you say "fixation and freezing and -80C", are you talking about antibody stocks themselves, or are you asking whether you can stain cells, fix, and then freeze them?
Re: Fixing/Freezing Metals
I meant during the staining procedure. We are trying to develop protocols that include a freezing step after surface staining of cells and fixation of cells.
Re: Fixing/Freezing Metals
I have not done this, personally.
I do know of a few cases with particularly biohazardous samples where people have done the antibody staining of live cells, then fixed (so no longer infectious) and shipped on dry ice. The results that I've seen are at least ok. You just have to make sure your cells are firmly fixed so the freeze-thaw doesn't damage them.
To my knowledge, the polymers and chelated metal ions are stable to freezing and thawing.
I do know of a few cases with particularly biohazardous samples where people have done the antibody staining of live cells, then fixed (so no longer infectious) and shipped on dry ice. The results that I've seen are at least ok. You just have to make sure your cells are firmly fixed so the freeze-thaw doesn't damage them.
To my knowledge, the polymers and chelated metal ions are stable to freezing and thawing.
Re: Fixing/Freezing Metals
Great, and what reagents/conditions would you describe as a firm fixation? We're currently using freshly made 200µl 2% PFA for 30 mins on ice for less than 6 million leukocytes.
Re: Fixing/Freezing Metals
We typically do 2% PFA in PBS (freshly-made from 16% methanol-free stock open less than 1 month) overnight at 4C. This is convenient for our workflow on 1-2 million PBMCs.
2% PFA for 15-20min at room temp can also work, but I don't personally use that workflow.
The 1-month limit is critical: almost everyone I know who's done CyTOF work has lost samples/plates when the PFA starts to go off, and the cells lyse in the MilliQ water washes and resuspension at the end of the protocol. If the cells are fixed well enough to survive MilliQ water, they should be good enough for freezing in PBS/FACS or some other buffer. Heck, even freezing *in* 2% PFA/PBS might be a good way to do it.
2% PFA for 15-20min at room temp can also work, but I don't personally use that workflow.
The 1-month limit is critical: almost everyone I know who's done CyTOF work has lost samples/plates when the PFA starts to go off, and the cells lyse in the MilliQ water washes and resuspension at the end of the protocol. If the cells are fixed well enough to survive MilliQ water, they should be good enough for freezing in PBS/FACS or some other buffer. Heck, even freezing *in* 2% PFA/PBS might be a good way to do it.
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