Mostly ELISA is used to quantify cytokines in cell supernatants but it also possible to do by flow cytometry for determination of cytokine producing cells (Elispot works as well).

It is critical to block secretion of cytokines during the final hours of the experiment. This is done by protein transport inhibitors like Monensin or Brefeldin A.

After harvesting, you need to fix and permeabilize your cells so that your antibodies could bind to their intracellular targets.

I would recommend using directly labeled antibodies. You may also need some cellular markers as CD4, CD19, CD14 etc. in case you are working with a heterogeneous cell population.

If you want to do double/triple stainings (detection of both IL6 and IL10 producing cells for example), you need to choose three different fluorochrome.

I use antibodies from eBiosciences and here is a detailed intracellular staining protocol from their website.

http://media.ebioscience.com/data/pdf/best-protocols/staining-intracellular-antigens-for-flow-cytometry.pdf

There are kits (CBA kits or Cytometric Bead Array) that may be purchased from BD that measure cytokine concentrations using cell culture supernatants. They use beads of a known size and fluoresence conjugated with a specific antibody. You can assay for up to 6  analytes with one flow cytometry run which justifies the high-ish cost of the kit. Look here: https://www.bdbiosciences.com/nvCategory.jsp?action=SELECT&form=formTree_catBean&item=745475

Unfortunately, from your question it is not clear whether you want to measure which cells produce cytokines (with intracellular flow cytometry) or multiple cytokines at once in the supernatant from cell cultures (using multiplex technology). In either way, I do not think you'll be able to use your monoclonal antibodies, but detection reagents needed for specific assay. 

Regarding intracellular flow cytometry for cytokines - check BD, eBioscience, Biolegend or any other provider website for the antibodies and reagents they are offering. I would recommend that you decide for one company and follow their protocol with their reagents. I have successfully used eBioscience reagents in my experiments. 

For multiplex technology, beside CBA kits from BD that Emily Turner-Brannen already suggested, I know that Biolegend offers Legendplex kits (but haven't used any of them). 

To quantify multiple cytokines in one sample, Luminex is a more mature technology.  If you have access to a luminex system, it is the better option.  The luminex system is built as a plate-reading flow cytometer specific for luminex beads.  Luminex has licensed its technology to any company that wanted it so you can find luminex kits at biorad, emd millipore, Life technologies, etc.  If you don't have access to a luminex system then to my knowledge CBA is the only way to go. 

If you want to know which cells secrete the cytokine, an interesting option is to detect cells that actually secrete (and not only produce and store) the cytokine.  You can use Miltenyi Biotec secretion assays to do that.

Good luck! 

http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-reagents/cytokine-producing-cells.aspx

http://www.luminexcorp.com/

Hi James,

I agree with Emily's response above - Cytometric Bead Arrays are the way to go by flow cytometry.  At iQ Biosciences, we routinely perform cytokine release assays using in-vitro generated supernatants for functional & safety purposes.  The benefits in using the CBA methods are significant (validated kit, perfect for limited sample volumes, multiple cytokines in ~ 4 hours).  

To answer your questions below:

1. You could use your own antibodies if you want to perform intracellular cytokine staining which would give you data in terms of percentages but not quantitative numbers (pg/ml) like the CBA method.

2. You can run the CBA on almost any FACS machine with two lasers so the low-tech approach would be using the FACS Calibur,

The caveat to the CBA method is that you will need to purchase the kit and software from BD Pharmingen in order to perform this.  If you think you will perform this regularly, it is worth the investment.  Otherwise, you may simply wish to work with contract research organizations that can perform this for you at a reasonable cost with fast turn around time.

I've pasted in some links to give you more background information as well as what representative data you may obtain as well.  If you do decide to proceed with the CBA, please email me at [email protected] and I can share with you a refined protocol that we use here at iQ Biosciences internally.  It reduces the volume of the reaction and still gives you solid results while saving you 50% on cost, etc.

Hope the answer is helpful.

Omar

http://www.iqbiosciences.com/bioservices/safety_assessment/cytokine-release-assay/

http://www.iqbiosciences.com/cytokine-storms-brewing-pt-1/

http://www.bdbiosciences.com/research/cytometricbeadarray/formats/index.jsp