I would also recommend serum or plasma and look for maximal response to decide for a specific time frame for analysis, like Marcin suggested. For spleen or lymph nodes I used lymphocyte isolation and in vitro re-stimulation with protein antigen for 48-72 h. Afterwards take the supernatant and immediatly measure via ELISA or freeze supernatants for later analysis. Take care especially for IL-4, in my experience the levels rapidly decline and than might be under detection limit of the ELISA. However, of course this depends on the experiments and in vitro stimulation conditions. Better freeze at -80°C and don't store longer than several months until analysis. Another option like already mentioned above are flow cytometry based assays e.g. CBA cytometric bead assays of serum or cell culture supernatants or intracellular cytokine staining of isolated cells. Or have you thought about ELISPOT...

There are many options ;) good luck for your experiments!

The best way is to use the Luminex instrument. Check it up. It is a multiplex platform where you can easily read these cytokines, together with many other. It is faster and cheaper than ELISA and you use less sample. The only limitation is that you need to find a group whi has the instrument.

Multiplexing can also be done with flow cytometry kits, e.g. CBA from BD Biosciences or similar, cheaper from eBiosciences. They both use small amounts of input sample and show little day-to-day variation.

Good luck!

Second to the advises of using Luminex multiplex. We have one machine in our group and it is easy to use, very fast, and very sensitive. As regarding to where and when to get your samples, you need to do them all to understand the variations with time and locations in order to optimize your protocol. Otherwise try to find publications doing similar study and check where and when did they collect their samples.

ELISA (multiplex) for the plasma and flow cytometry for cell analysis. You can culture cells and measure the secretion by ELISA. Alternatively go with Johan's suggestion us flow cytometry instead of an ELISA (FlowCytomix™ Multiplex Kits: http://www.ebioscience.com/product-line/flowcytomix/flowcytomix-multiplex-kits.htm

Our lab has used the Luminex system, ELISA and R&D systems spot blot cytokine array. We have measured cytokine secretion from both cells and sera. Regarding where, I suggest sera, spleen and tissue of interest. Our collaborators (Dr. Chris Jolly) have detected differential secretion of IL-17, IL-10, IFN and IL-4 from activated T-cells isolated from mice in as little as 24 hours. As Jiahn stated, I would use publications as a starting point for timing and location to optimize your studies.

I would test serum levels of your cytokines, I reckon that you injected your antigen intravenuously?

For intravenuous cytokine induction it depends on the stimulus but you should see a response between 1 and 4 hours if you have a good activation (IL-4 hould be earlier thant the other cytokines) but you can always try different time points.

You can always try different time points.

From where is it advisable to sample cytokines for ELISA - plasma, lymph node or spleen?

From plasma!

Can be compared with the parameters from lymph node and spleen.

Thanks for the quick answers - i will take blood sample for plasma at different time point up to 48h after boost injection, but of course for spleen or lymph - it is only at one time point.

(the antigen injected i.m)

I think this is the right decision. Тhe antigen injected i.m.

If without the adjuvant, the antigen is preferably administered intravenously or intraperitoneally.

Tal, my 2 cents: do the plasma profile first, then select a time-point where you have them all up (best case scenario) or at least some of them, and use it as sacrifice time-point to get the organs for comparative analysis. Would not hurt if you kill mice at this "peak" time-point but also at 1 earlier time-point that precedes the observable plasma peak. Due to compartmentalization of cytokine production/secretion you may actually see some interesting things there. :-) wish you that.

I agree with Dr. Marcin Osuchowski.

I would also recommend serum or plasma and look for maximal response to decide for a specific time frame for analysis, like Marcin suggested. For spleen or lymph nodes I used lymphocyte isolation and in vitro re-stimulation with protein antigen for 48-72 h. Afterwards take the supernatant and immediatly measure via ELISA or freeze supernatants for later analysis. Take care especially for IL-4, in my experience the levels rapidly decline and than might be under detection limit of the ELISA. However, of course this depends on the experiments and in vitro stimulation conditions. Better freeze at -80°C and don't store longer than several months until analysis. Another option like already mentioned above are flow cytometry based assays e.g. CBA cytometric bead assays of serum or cell culture supernatants or intracellular cytokine staining of isolated cells. Or have you thought about ELISPOT...

There are many options ;) good luck for your experiments!