labeling of cytokine releasing cells - flow cytometry (Sep/22/2006 )

Hello,

I saw once a flyer about a kit allowing to label the cell that is secreting a cytokine and you can analyse it on a flow cytometer, or am I dreaming?
there is a molecule in this kit that binds to the cell membrane and binds also the cytokine or an antibody directed against the cytokine.

do you see what I mean?
have you ever tried this kit?

Thanks in advance

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cytometric bead assay "cba" by bd biosciences...it only detects the cytokines though...

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this is usefull to quantify several cytokines in the supernatant.
It's not really what I wanted.
thanks for your answer anyway.

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There's ELIspot where you detect cytokine secreting cells in a culture plate coated with a cytokine capture antibody. After the detection steps you get spots where the cytokine secreting cells were.

There's also intracellular cytokine staining protocols where you detect the cells expressing cytokines. You can treat the cells (if they're in culture) so they don't secrete the cytokine (using Golgiplug I think it stops endosomal transport) then permeabilise the cells and detect by flow cytometry with a PE-/FITC-labelled antibody. I think BD must have the protocols on the website.

Ceri

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you're right Ceri, this is how I will do, block cytokine release with Brefeldin A, and fix and permeabilise the cells.
It seems that the kit I was thinking about is unfindable. It could have been interesting to use to sort the cells, but maybe it was not efficient enough (or maybe I was really dreaming )

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Hi Missele,
Sorry I see what you meant now. You want to sort viable cells expressing cytokines and the kit you describe vaguely rings a bell. I've now idea who made it though. You could try searching the internet for it.

Could you initially see which cells are expressing the cytokine by intracellular flow cytometry and look at specific cell surface markers to see if it's specific subsets then sort for these surface markers?

All the best,
Ceri

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so maybe I was not dreaming
I will continue to search (I was thinking about this, while I need to label cells for cytokine for an other purpose) usually it's when you are not looking for something that you find it ! so I will not stres too much and permeabilize the cells for this round.

(unfortuately the cells that are secreting or not have the same phenotype for the known markers)

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I think I know what you're talking about. I used this kit from Miltenyi Biotec to measure the amount of interferon gamma secreted by a cell. It uses a bivalent antibody, one end that binds to the surface of the cell, and the other end to bind to the cytokine of interest. Usually it comes in several different colours, which you can then optimize with your assay for flow cytometric analysis

Check out their website: http://www.miltenyibiotec.com

You'll find more information on this, and the product I used was 130-054-202

I hope this helps

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