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Longterm storage of phospho samples in methanol

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Posts: 3

Joined: Mon Sep 10, 2018 1:49 pm

Post Thu Mar 19, 2020 4:17 am

Longterm storage of phospho samples in methanol

Hello CyTOF community,

We have been conducting a large multi-week phosphoCyTOF experiment in our lab. We were planning on thawing 14 plates worth of samples, with each plate being run on a different day. We are stimulating, fixing, barcoding, extracellular staining the cells on the day we thaw that plate’s samples, and storing them in methanol at -80. The hope was that once all 14 plates of samples were stored in methanol, we would proceed and perform intracellular staining on all the samples on the same day, to avoid batch effects by using the same intracellular antibody cocktail. We have run half the samples, but in light of the recent COVID19 crisis around the world and concerns with running experiments with many people in the same room, we are unsure about when we will continue these assays. The samples that have already been run are in methanol at -80, but according to https://www.ncbi.nlm.nih.gov/pubmed/27798817, there is some concern about keeping in samples in methanol for over a month, namely that your cell yield will decrease and intracellular marker staining may be affected. It seems like it may not advisable to transfer those samples to an FBS/10% DMSO solution before intracellular staining.
I’m curious about other others’ experiences with storing samples and staining.
We would prefer to have the same intracellular antibody cocktail for all the samples, but would keeping them in methanol for potentially another month or longer create a much worse batch effect than just staining the samples we have now separately from the samples that have not been run?
Our intracellular markers are phosphomarkers, but the intracellular staining from the paper, which was affected by prolonged methanol storage, were mainly cytokines. Does anyone know whether this trend applies for phosphomarkers as well?

Thanks for the help! :)
Darius M.



Posts: 4

Joined: Thu Feb 28, 2019 2:43 am

Post Sat Mar 21, 2020 8:12 pm

Re: Longterm storage of phospho samples in methanol

One way or another, you will have to correct for batch effects during post-processing and analysis.

Choi et al describes a way to do this relatively simply: https://www.jacionline.org/article/S0091-6749(17)30586-9/fulltext. There are other methods, however, if you running the frozen control samples alongside your experimental samples. (ie misc R packages)

So long as you're correcting for batch effects I don't anticipate either being a terrible problem. I will say, however, that keeping them all in methanol will standardize the procedure and mitigate inter-sample differences.

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