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Live cell barcoding and intracellular staining

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marthabrainard1

Contributor

Posts: 20

Joined: Wed Aug 25, 2021 2:10 pm

Post Wed Sep 22, 2021 5:19 pm

Live cell barcoding and intracellular staining

Hi! I am curious about implementing a live-cell barcoding protocol and was wondering if anyone has any suggestions about the fix/perm necessary for intracellular staining. Some of the questions I have are below:

1. Do you skip palladium barcoding fix/perm steps or do these incubations somehow prep the cells for the harsher eBio fix/perm? We wouldn't be adding palladium barcodes, but are wondering if anyone has QC'd getting rid of these steps and how that affects sample quality and intracellular staining
2. Have you been able to shorten the eBio fix/perm incubation
3. Have you used any other nuclear staining/transcription factor kits other than eBio
4. Have you skipped the palladium barcoding fix/perm steps and used a less harsh fix/perm (BD cytofix/cytoperm)
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CRStevens

Master

Posts: 60

Joined: Thu Jul 17, 2014 5:07 pm

Post Wed Sep 22, 2021 5:47 pm

Re: Live cell barcoding and intracellular staining

I have done some work with the Cd-CD45 LCB system and will caution that in my whole blood system the Cd doesn't play well with some Lyse/Fix buffers. It is unclear what about those buffers caused the lack of staining, but I would definitely try a trial run with your buffers before running precious samples.

I also can say that the Cd-CD45 is very dim and needs to be titered up. Don't assume you can use 1uL per million cells (for whole blood we had to use 3-4uL / 100uL of blood, which is about 1million). The Pt based CD45's seem to be much better one both fronts of having better signal and being a little more resistant to buffer effects.

For intracellular staining you will not need to use the Pd-barcode fixation first if you are not doing Pd-based barcoding, just stain your samples with the LBC reagent as normal and proceed to your Fix/Perm protocol of choosing, Again never have tested the Cd or Pt labeled CD45's with these buffers, so please do so before proceeding.

Would also love to hear about your results from running this as well! Good luck.
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marthabrainard1

Contributor

Posts: 20

Joined: Wed Aug 25, 2021 2:10 pm

Post Wed Sep 22, 2021 6:03 pm

Re: Live cell barcoding and intracellular staining

Thank you, good to know that the Pt based CD45s are less dim. I'll see how it works with our protocol!
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jobagins

Participant

Posts: 13

Joined: Thu Oct 22, 2020 6:45 pm

Post Wed Sep 22, 2021 6:11 pm

Re: Live cell barcoding and intracellular staining

MB we did some testing

- we barcode CD45 and then regular surface, fix/perm, intra, fix, Ir.
- eBio - how long you do it? 20min is enough
-yes we did with worse outcomes
-yes

We should discuss in person because I miss you
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marthabrainard1

Contributor

Posts: 20

Joined: Wed Aug 25, 2021 2:10 pm

Post Thu Sep 23, 2021 12:39 pm

Re: Live cell barcoding and intracellular staining

Hi Joanna! Thanks for the response-we will have to try 20 min incubation

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