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removing debris

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klavine

Participant

Posts: 2

Joined: Wed Sep 07, 2016 2:51 am

Post Wed Sep 07, 2016 5:22 pm

removing debris

We have been struggling with strategies to remove debris from our sample. Currently, our samples have 95% debris and 5% cells. Is it possible to include a fluorescent marker (such as CD45-PE) with your CyTOF antibody panel and purify on a cell sorter prior to running through the CyTOF instrument? I was not sure whether heavy metal conjugates would be corrupted during traditional cell sorting or damage the instrument.
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HRsumatoh

Participant

Posts: 8

Joined: Fri Oct 24, 2014 2:15 am

Location: Singapore

Post Thu Sep 08, 2016 2:32 am

Re: removing debris

Hi klavine

We have adopted a strategy where we would stained the cells with fluorophore-conjugated abs and put them in the sorter to enrich the cells of interest (e.g. Live, CD45+ Bcell- cells). After which the cells are stained with your Cytof ab panel. We did not see any interference in the ab binding if you incorporate, say for example, CD45-metal tagged ab with an already-stained sample with CD45-fluorophore tagged ab. We have been adopting this strategy for some time now and it works.

Cheers
Rizal
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mleipold

Guru

Posts: 1778

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Sep 08, 2016 6:55 pm

Re: removing debris

Hi klavine,

A few questions:

1. What type of sample are you running? PBMCs, WB, bone marrow, disaggregated tissue, BAL, etc?
2. What type of assay are you running? surface phenotyping, ICS, phosphoflow?
3. Do you have example data from your analysis of an affected sample? Do you have screen shots of the Rain Plot while your samples are running? Ideally, both Marker mode and TOF mode.


Mike
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klavine

Participant

Posts: 2

Joined: Wed Sep 07, 2016 2:51 am

Post Thu Sep 08, 2016 8:55 pm

Re: removing debris

Hi Mike,

We are using cardiac tissue digestions and are pursuing both surface and intracellular staining including phosphoflow. Our protocol is optimized for standard flow cytometry, howver we have not yet run any CyTOF experiments. I was thinking of using CD45-PE to isolate immune cells from the debris.

Thank you for your help.
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mleipold

Guru

Posts: 1778

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Sep 08, 2016 9:26 pm

Re: removing debris

Hi klavine,

Some people have used Miltenyi's dead cell removal kit to help get rid of debris: viewtopic.php?f=4&t=461&p=1471&hilit=dead+cell+removal#p1471

http://www.miltenyibiotec.com/en/produc ... l-kit.aspx

I think I've heard of other people (Adeeb?) using it as well. The Cytoforum link above noted that it caused some distortion in some cell subsets, but that was in whole-blood: your sample might be fine.


Mike

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