It depends on what kind of equipments you have! With a plate spectrophotometer you could go with ELISA, if you have a plate fluorescence reader, go with alphaLISA, if you have a luminex machine and a lot of money go with xMAP kits, if you have a two-laser (488/633) flow cytometer you could use BD cytometric bead arrays, you could also perform dot blots, etc.

With more information on what you equipments you have and and idea of the targets you seek, we may be able to give you better information or specific protocols. Cheers! :)

I think, to start just identify some of the key cytokine you want to screed and do a standard ELISA. A lot of compagnies offer ELISA kit. YOu can also use flow cytometry with fixed cells to do intracellular staining of the cytokine. YOu will find the protocols easly on the net for that.

If you have a flow cytometer, bead arrays are particularly of value when you have a small sample. It allows the simultaneous detection of up to 40 cytokines. Depending on the manufacturer, you can find different sets that allow detection of most common cytokines for Th1, Th2 or Th17 responses. Some manufacturers may even offer customized sets for detecting less common cytokines, but it takes more time to standardize.

We are doing similar experiments with BEAS-2B cells using RT-PCR which is very straight forward if you have access to an icycler

Hi Stefan!

You want to detect protein, so I recomend sandwich ELISA. I'm used to the Biolegend's kits for mice. They work pretty well and are less expensive than BD's or R&D, for instance. But you need to test for your human source. Try to ask for samples to test it.

Good luck.

Can use cytokines array methods, which could be in ELISA format but quite expensive. If flow cytometry is available at your laboratory, can consider flow cytomix assay which can detect up to 20 cytokines in one go and only require 10-20 ul supernatant. Cost effective

Thanks that is a good tip. I have been using the R&D kits and making my own plates for cell exposures to dusts. going to check out biolegend.

It really depend on the number of cytokines you want to detect. up to three different ELISA is the best and the cheapest method. I recommend the use of Luminex methodology for measuring more than 3 and up to 50, it's quite expensive but less than 50 ELISAS. Furthermore, the range of detection of this technique is one log more than ELISA. You can measure up to 50 cytokines in only 50microl... take a look of our work in serum and synovial fluid

Article Cytokines profiling by multiplex analysis in experimental ar...

Dear Stefan,

It depends how many cells you have seeded into the wells ( 6, 12, 24 or 96 wells) and what cytokines you want to measure. For example, IL-8 and IL6 after 12 or 24 hours of stimulation, I need to dilute the supernatants many folds to fit in the standard curve of ELISA, TNF-alpha or other cytokines need to measure in neat, then you can use multiplex system to measure or screen other cytokines of your interest depends on how much supernatant you got. I am normally centrifuge the supernatants to remove loose cells before I stored them.

It will depend in what equipment you can have access and how many molecules you want to assay. Multiplex analysis with a Luminex equipment is very convenient if you are planning to assay several molecules. I have very good results with this technique. You may find interesting my paper on Cytokine journal:

"Cytokine responses by conjunctival epithelial cells: An in vitro model of ocular inflammation  "

Cytokine, Volume 44, Issue 1, October 2008, Pages 160-167

Amalia Enríquez-de-Salamanca, Virginia Calder, Jianping Gao, Grazyna Galatowicz, Carmen García-Vázquez, Itziar Fernández, Michael E. Stern, Yolanda Diebold, Margarita Calonge  

If you have not this technique/equipment available, there are also several commercial ELISAS that you can use.

Hi,Stefan Bidula

I used human cytokine/chemokine Milliplex map kit (Millipore,Billerica,MA). It worked well.

It is good to use elisa kits as R& D kits,invitrogen,BIOMED SYSTEM,and flow cytometry etc for protein expression and for mRNA Quantification by real time pcr.

There is a laboratory in Framingham, MA which I have visited and has the best expertise I have yet seen in the analysis of cytokines. You should Google SBH Sciences for contact information and speak to Raphael Nir, the Laboratory Director,

for guidance.

Thank you everybody for your suggestions and help. You've left me with a lot more options

I use the CBA kit from BD. It works fine. Gives me all the data I need with 50ul of supernatant but you need to have a flow cytometer.

I think ELISA kits (Biolegend)) may be helpful, which asks for few sample and is cost effective.

Hi Stefan,

May be you may also find useful this multi-analyte ELISArrays from SABiosciences

http://sabiosciences.com/ELISA.php?utm_content=PR120919+ELISArray_Int&utm_campaign=PR120919+ELISArray&utm_source=iPost&utm_medium=email

Regards