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Fixation and Ir Intercalactor staining

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LouisS

Participant

Posts: 2

Joined: Mon Jan 27, 2020 12:48 pm

Post Fri Jan 31, 2020 10:34 am

Fixation and Ir Intercalactor staining

Hej all,
I am totally new to CyTOF and we are setting up our first panel. I have a few questions about the general protocol and how we could shorten/combine things. I guess a lot of users here have tried these things. We are running the Helios system.

1. Has anybody tried to stain Ir Cell-ID at the same time as the intracellular antibodies in the Perm buffer of the FoxP3 kit?
2. Could you stain Ir Cell-ID in “% or 4% fixing buffer for 30 min after you have used the FoxP3 staining kit?
3. There are a lot of different protocols fixing with either 2% or 4 % PFA. Is there any disadvantage/advantage of one over the other?
4. What about leaving the cells in 4% PFA with Ir Cell-ID overnight in the fridge? Do you lose many of the cells or is it okay?
5. Any experience with staining CD45 barcoding while staining with Rhodium-103 for live/dead discrimination?

Thanks for your help!
Cheers, Louis
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Jan 31, 2020 5:11 pm

Re: Fixation and Ir Intercalactor staining

Hi Louis,

It's reasonably common to stain Ir along with Fix after intracellular stain (#2).

I can't comment on 2% vs 4% PFA (#3): honestly, this is something you're going to have to test for *your* system to see what ensures adequate fixation. Please remember, if your cells are not fixed sufficiently, they are likely to bust during the final washes, causing poor cell recovery and a lot of streaking.

Regarding #4: you can definitely leave the cells in PFA/PBS overnight with Ir. However, you often need to lower the Ir concentration to avoid overstaining......rather than 1:2000 for same-day staining, it is more common to use 1:5000 or even 1:10,000 dilution. Again, you're going to have to test out what works for your system.

Regarding #5: formally, you could do this (similar to the Lyophilized tube staining that Fluidigm sells). However, my opinion is that you should stain Live-dead as *late* in the protocol as you can: cells can die *during* staining, and if you do live-dead at the beginning, you will *not* be counting the dead cells completely. As such, I personally think that you should do live-dead as a step immediately before your fixation in order to count them properly.


Mike
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LouisS

Participant

Posts: 2

Joined: Mon Jan 27, 2020 12:48 pm

Post Mon Feb 03, 2020 8:56 am

Re: Fixation and Ir Intercalactor staining

Thanks for your reply Mike! That will already help us to decide what to try :)

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