Mouse live cell barcoding with CD45

[Osiris]

Hi there,

Has anyone else tried to label mouse CD45 with pSCN-Palladium isotopes for live cell barcoding?

I followed the simple protocol from Mei et al., Barcoding of Live Human Peripheral Blood Mononuclear Cells for Multiplexed Mass Cytometry, 2015 (http://www.ncbi.nlm.nih.gov/pubmed/25609839) published recently. However I find that the antibody gets dried up in an orange precipitate after the incubation period and there's no signal when I stain the cells. This doesn't happen with the human antibody. I can shorten the incubation period and spin at a lower speed for the subsequent washes but I just wondered whether anybody has had a similar experience and might have some ideas or tips?

Thanks!

Dunja

[henkmei]

Hi Dunja,

I am excited to see that you're trying CD45 barcoding for murine cells. Until now, I did not run into the issue you describe....; "precipitate" does not sound good to me. I remember that we once wished to upscale our Ab production and allowed for a DMSO concentration (DMSO coming from the ITCB -EDTA) of 10% rather than the 2% described in the paper in the conjugation reaction. With 10% DMSO we ended up with at least lower staining signal and about half the protein recovery determined by Nanodrop as compared to the 2% DMSO conjugation, but I can't remember a precipitation.

If you don't mind sharing details of your ITCB/loading & Ab conjugation experiment (here or by email) I could have a look at it.

Best
Henrik

[Osiris]

Hi Henrik,

Thanks for the reply! I sent you a PM but it didn't appear in my sent messages so I'm not sure if you got it...?

Best,
Dunja

[henkmei]

Hi Dunja,
I didn't get any PM, my inbox is empty. You can email me to hemei@stanford.edu.

Best
Henrik

[Pat1999h]

Dear Henrik,


I am interested if you could solve the problem with CD45 barcoding in mice.

Sincerly


Patrick Hölker