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low event rate using methanol permeabilization

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lilaczhang

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Joined: Fri Feb 15, 2019 3:29 pm

Post Wed Feb 20, 2019 4:14 pm

low event rate using methanol permeabilization

I compared ebioscience fix/perm kit and methanol protocol. The cell count number after staining is similar for both. However, the event rate in methanol group is very low, around 1/3 of ebioscience kit or even lower in same cell concentration. Does anyone know how to fix this problem? Thanks!
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kunicki

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Joined: Thu Apr 13, 2017 8:46 pm

Post Wed Feb 20, 2019 10:53 pm

Re: low event rate using methanol permeabilization

Sounds like a cell stability issue during acquisition. Is the fixation between Fix/Perm and MeOH protocols identical? There are protocols posted in the forum where experts have suggested adding additional fixation steps, or slightly increasing the %PFA.
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JaromirMikes

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Joined: Tue Sep 29, 2015 7:19 am

Post Thu Feb 21, 2019 11:43 pm

Re: low event rate using methanol permeabilization

methanol protocol is relatively aggressive and destabilises cellular structures, therefore cells fall apart in last steps of washing with diH2O and it goes on when the samples are waiting in queue. As this process might be non-stochastic, there's a high risk that the acquired data are biased. I presume you count cells after staining but before washing in diH2O, right?
As mentioned by Matthew, cell might need another step of stabilisation or a different protocol.
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sedejong

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Joined: Wed Mar 04, 2015 3:42 pm

Post Fri Feb 22, 2019 12:54 am

Re: low event rate using methanol permeabilization

I have done a similar comparison between the eBioscience FoxP3/TF staining buffer set and methanol. I did not see a difference in cell counts after staining and in event rate. The methanol-treated cells actually seemed to be sticking less to the CyTOF capillaries and, probably because of that, I even ended up with slightly more acquired cells with methanol.

So I don't think methanol itself is to blame, but maybe our protocols are different somewhere else? I did a fresh fixation as recommended by Fluidigm after my nuclear staining and before the DNA stain. I also did this fresh fix after my surface staining and before the methanol incubation. So I do this: Viability stain > surface stain > fresh fix > nuclear stain > fresh fix > DNA stain. If you weren't doing so already, maybe you can do this too and see whether it helps?
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lilaczhang

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Joined: Fri Feb 15, 2019 3:29 pm

Post Fri Feb 22, 2019 3:04 am

Re: low event rate using methanol permeabilization

Thanks all for the very helpful information.

@sedejong,
I have similar protocol as yours: viability staining, surface staining, 1.6%PFA fix, ice-cold methanol, intracellular staining, 1.6%PFA fresh fix, intercalator

I just made some small changes on the protocol yesterday and it worked well on the Helios today. it gave me similar cell count and event rate as ebioscience kit.
Small changes:
1. loose the pellet by vortexing, then add ice-cold methanol without pipetting or vortexing (I did pipetting or vortexing before)
2. pellet the cells after 2nd wash in dH2O, and run the samples immediately, don't leave the cells in pellet too long.

I think the 1st one might be critical and not sure how much help the 2nd one did.

@JaromirMikes:
I count the cells after 1st wash with dH2O, and then pellet the cells in 2nd wash with dH2O. I usually wash and pellet all my samples (4-6 samples) at once and leave the cells in pellet with small amount of dH2O until collecting data. It works for ebioscience or Fluidigm protocol. Do you know how long the cell pellet can stay in the dH2O for methanol protocol? Samples need to wash one by one or can be washed all together?

If the cells fall apart in dH2O, how long can you record the data for one sample to avoid the bias?

Thanks again!
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JaromirMikes

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Joined: Tue Sep 29, 2015 7:19 am

Post Sat Feb 23, 2019 9:49 pm

Re: low event rate using methanol permeabilization

Both mentioned steps are important as they might have cumulative effect. For example some protocols for fixation of cells with alcohols for cell cycle analysis (it's analogous to perm step in your protocol) recommend vortexing during alcohol addition but it mostly says "a gentle vortexing". I did some testing years ago and confirmed that mechanical stress during fixation with ethanol can be critical for larger cells or trypsinized adherent cells. Suspension cell lines were less sensitive. Therefore, experience from lab to lab might differ. E.g., there are differences in sample types. Whole blood samples containing neutrophils are more sensitive to processing steps than PBMCs. And a quality of the first fixation step is critical, too. Especially when the samples is later challenged with multiple perm steps.
Fixation step after methanol permeabilisation definitely helps to increase sample stability, though it might need some changes. Cells fixed with formaldehyde and processed without alcohol permeabilisation are more robust so the optimal protocol will differ. How long is your last fix step?
In case you can't skip the methanol perm step my recommendation is:
1. elongate the fixation step after methanol perm but...
2. still minimise the time in diH2O.
No matter how intensively you post-fix the methanol-permed cells you can't expect the same stability as the changes to cellular structures are irreversible.
Did you try to skip the methanol permeabilisation?

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