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CD45 vs Palladium barcoding

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Joined: Wed Nov 20, 2019 5:20 pm

Post Sun May 31, 2020 3:04 am

CD45 vs Palladium barcoding

Dear Forum, which of these: CD45 or palladium barcoding do you prefer and why?



Posts: 113

Joined: Mon Oct 31, 2016 6:26 pm

Post Sun May 31, 2020 3:05 am

Re: CD45 vs Palladium barcoding

Plugin to the barcoding survey I held last year:




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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Jun 01, 2020 12:14 am

Re: CD45 vs Palladium barcoding

Hi Nameera,

One of the big questions you have to ask yourself when you're barcoding is whether you're doing your main stainings on Live cells or on Fixed cells.

The Fluidigm barcoding (I assume that's what you meant by "Pd barcoding") or the Bodenmiller/Zunder small molecule barcoding both require perming the cell. Therefore, this can *only* be done on Fixed cells. This was first developed for Phosphoflow assays, where all the samples would be fixed at the end of the phospho stim. You can do a gentle perm like Greg Behbehani's paper (https://doi.org/10.1002/cyto.a.22573) for barcoding, in case any of your surface antibodies are sensitive to harsher perms like Methanol.

The CD45 (or CD298 or b2m, or any other antibody- or affinity-reagent-based) barcoding does *not* have this requirement: it was developed for use on Live cells. However, many of the epitopes like CD45 are generally PFA-stable, so many or most of the antibody barcodes can be used on Fixed cells too.

Therefore, if your samples are immediately fixed like SmartTube on fresh whole blood, or an assay like phosphoflow, then your antibodies are probably fixation-stable. However, if you're doing a surface phenotyping assay or ICS assay where you stain live cells, you're going to have to test whether your epitopes give decent staining after fix.

Or, do the surface staining *separately*, then barcode, then combine for any intracellular, Ir, and then running.


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