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Antibody titration on barcoded samples

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marionkonig

Participant

Posts: 2

Joined: Wed Mar 20, 2019 11:16 am

Post Thu Oct 29, 2020 3:29 pm

Antibody titration on barcoded samples

Hi all,

I am planning a big experiment in which I will barcode and combine 22 PBMC samples, a total of about 70 million cells.
Do any of you have suggestions for how much to increase the staining volume and/or the titrations? I did the titrations on about 3 million cells in 100ul

Previously I combined 20 million cells and increased the titrations 1.33x and the staining volume to a total of 200ul, which worked fine.
For another experiment with 50 million cells the signal intensities already seemed to be significantly lower, so I'm wondering how others deal with this

Thanks!

Marion
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mleipold

Guru

Posts: 2640

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Nov 02, 2020 4:09 pm

Re: Antibody titration on barcoded samples

Hi Marion,

Short answer: I think you're going to have to just test it on 50-70M PBMCs. I realize that it's "expensive", but I don't know of any rule of thumb for converting a 500K-3M experiment into a 20M experiment into a 70M experiment.

In my own experience going from standard 1M staining to a barcoded 8-10M staining, there wasn't a smooth relationship. Relative to 10 individual samples of 1M, some antibodies could be used at 0.5x proportion (eg, calculate for 5M when staining 10M BC), others were at 0.75x (calculate 7M when staining 10M BC), and a few that had to be used at full relative titer (calculate 10M when staining 10M BC).

As expensive as it is to do a test at 50-70M, it's still cheaper than your experiment not being interpretable.


Mike
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AndrewD

Participant

Posts: 10

Joined: Tue Feb 20, 2018 7:18 pm

Post Tue Nov 03, 2020 11:03 am

Re: Antibody titration on barcoded samples

Hi Marion,

I agree with mike, there is no easy way to work out how to scale the antibody titre. The signal reduces due to saturation of the antibody and this will depend upon the abundance of the antigens in your samples and the efficiency of the antibody binding.

Just be aware that when performing a large barcoded experiment with 70M cells, you will not be able to run this number of cells through the mass cytometer in one day; even 500 events/sec for 12hrs will only give ~20million events. It may be best to only wash the cells that you are immediately about to run through the CyTOF and leave the remaining cells in fixative until they are ran (maybe the following day). You should also tune the CyTOF every 4hrs during collection and then you can concatenate all FCS files together before debarcoding.

HTH
Andrew
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mleipold

Guru

Posts: 2640

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Nov 03, 2020 3:21 pm

Re: Antibody titration on barcoded samples

To elaborate on Andrew's comment about runtime:

You might want to consider freezing your sample in aliquots. We have done that for a few projects: stain up through and including Iridium, do a wash, then aliquot and freeze in FBS+10% DMSO. That way, you can thaw only the aliquot that you're planning to run that day.
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marionkonig

Participant

Posts: 2

Joined: Wed Mar 20, 2019 11:16 am

Post Thu Nov 05, 2020 11:46 am

Re: Antibody titration on barcoded samples

Thanks all for your replies!

For this time we decided to split our barcoded sample in multiple batches for the staining, since we didn't have time to test this all.
Of course this may cause some variation but at least we can pool them for the acquisition.

We froze our samples in multiple vials so that we can thaw and acquire 1 vial per day.

Marion

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