CyTOForum

Hello everyone.
I have started using CyTOF on endometrial samples but the barcodes I have been using for PBMCs (β2 microglobulin and CD298 unfortunately do not work great for it. Does anyone have any ideas on how best to barcode these samples, which include multiple cell types?
Kind regards,
Danai

Hi Danai,

Could oyu give us more information about your project? For example:

1. I assume since you're using the antibody-based barcoding approach that you're trying to barcode Live (unfixed) cells. Is that correct?
2. You mention "include multiple cell types": are you trying to barcode all your cells, or just immune cells?
3. One issue affecting antibody-based barcoding in particular is the overall sample quality: in particular, debris and/or dead cells can throw off your effective titer. When you do run these samples, are they nice punctate cell events, or is there a lot of streaking?


Mike

Thank you very much for your immediate response Mike.
My cells are Live and are fixed at the end of my protocol. Unfortunately, I am not just interested in immune cells. which creates a need for a more universal marker. I am removing dead cells prior to the analysis and the data seems fine otherwise (apart from the minimal barcode staining).
Best regards,
Danai

Hi Danai,

Removing dead cells during your analysis isn't exactly what I meant: by that point, they would have thrown off your CD298 and/or b2m titer. The primary way you'd see how this is affected is by looking at the Rain Plot during acquisition, and seeing how streaky the BC channels are (or, perhaps, all of your channels).

The other recommendation that I have is to talk with your SBio FAS about the TeMal barcode agents that can be used on both live cells or dead cells:
Mark Nitz's lab's original publication: viewtopic.php?f=10&t=1131
Christopher Tape's lab has been using them with success: https://doi.org/10.1038/s41596-021-00603-4


I know from talking with some of the SBio people at CYTO2023 and since that SBio is in preliminary (beta?) customer testing for this.


Mike

Thank you very much Mike