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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 :-)
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DAVID

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Post Thu Nov 28, 2013 9:08 am

Fixing/Freezing Metals

Are metal conjugated Abs stable after fixation and freezing at -80? Does anything happen to metals kept at low temperatures for a long time?
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mleipold

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Location: Stanford HIMC, CA, USA

Post Fri Dec 06, 2013 10:13 pm

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?
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DAVID

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Posts: 17

Joined: Tue Nov 26, 2013 1:55 pm

Post Thu Apr 03, 2014 2:44 pm

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.
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mleipold

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Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Apr 03, 2014 3:12 pm

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.
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DAVID

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Posts: 17

Joined: Tue Nov 26, 2013 1:55 pm

Post Thu Apr 03, 2014 3:35 pm

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.
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mleipold

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Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Apr 03, 2014 4:02 pm

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.

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