Dear Nguyen,

May be this Multi-Analyte ELISArray Kits helpful for you to measure several cytokines. You can find the details in this link

http://www.qiagen.com/products/catalog/assay-technologies/protein-and-cell-assays/multi-analyte-elisarray-kits?utm_content=QFDKPR141003JL+ELISArrays+with+Immunology_us&utm_campaign=QFDKPR1410+October+2014&utm_source=iPost&utm_medium=email&cmpid=QFDKPR141003JL+ELISArrays+with+Immunology_us&imm_mid=0c42eb

MSD Elisa. http://www.mesoscale.com/CatalogSystemWeb/WebRoot/landing_pages/immunogenicity.aspx

ELISA is a good method, as Mafalda points out but usually can only do one cytokine at a time (as the plates are coated with one specific antibody). If you are trying to measure several at a time, I recommend a cytometric bead array. It will cost you some money, and you will need access to a flow cytometry facility but it is definitely one of the gold standards.  You can find the kits here:

http://www.bdbiosciences.com/nvCategory.jsp?action=SELECT&form=formTree_catBean&item=745475

I do not know of anyway to measure 20-50 cytokines at once other that total proteomics through mass spectrometry. I know it works, but there is definitely a lot that needs to be worked out before you can get it to work quantitatively and reliably.

Good luck,

~Adam

Dear Nguyen,

May be this Multi-Analyte ELISArray Kits helpful for you to measure several cytokines. You can find the details in this link

http://www.qiagen.com/products/catalog/assay-technologies/protein-and-cell-assays/multi-analyte-elisarray-kits?utm_content=QFDKPR141003JL+ELISArrays+with+Immunology_us&utm_campaign=QFDKPR1410+October+2014&utm_source=iPost&utm_medium=email&cmpid=QFDKPR141003JL+ELISArrays+with+Immunology_us&imm_mid=0c42eb

with personal experience in this, I would like to suggest to use Millipore Multiplex/milliplex Kit. A little bit expensive but worth it. You can analyse 20-25 cytokines in one go with good data quality.

ref: http://157.93.252.5/catalogue/item/HCYTOMAG-60K

I have recently used a bead-based multiplex assay from Biorad. Quite cheap and very short bench processing time.

http://info.bio-rad.com/ww-Bio-Plex-Luminex-lp1.html?WT.srch=1&WT.mc_id=aw-pfd-EU-bioplex_luminex_gold&WT.knsh_id=379670b8-c532-26e9-2b8b-00003fcd0dc8&gclid=CjwKEAjw-8ihBRD2t9qT3NaW7igSJAD3_sNVMYhV_z61Pfctjb_I8iKqtLvNhsOnUDrSS0wH4EqvqRoCfyjw_wcB

Hi

You can use Cytokine Human Magnetic 30-Plex Panel for the Luminex Plataform. http://www.lifetechnologies.com/order/catalog/product/LHC6003M. Or Cytokine Mouse Magnetic 20-Plex Panel http://www.lifetechnologies.com/order/catalog/product/LMC0006M. You are not going to have enough sample to perform ELISAs for 20-50 cytokines (2-5 ml of sample). So, I think luminex could help you.

In my opinion antibody arrays can be an alternative to performing multiple elisa. You need around 200-500 uL of supernatant and you can find out the relative expression of proteins included in antibody array. I am attaching link to one such antibody array with this answer. Moreover, the readout is fairly simple and normal radiographs and scanners can be used to determine relative expression of proteins. Furthermore, this should be followed up with ELISA for absolute quantification of overe-xpressed cytokines.

http://www.rndsystems.com/ScientificPoster_ps_profiling_secreted_cytokines_from_th2_and_th17_cells_using_antibody_arrays.aspx

Hi

western blot could be used to detect the protein in supernatant and the amount of protein it could be quantified, as well. The problem is, cytokines normally have heavy post-translational alteration like heavy glycosylation. So it is hard to detect the right band and sometimes you may see several bands corresponding to your intended cytokines

ELISA is the most recommended technique here to measure the amount of protein.

Good luck

I suggest you using R&D’s Proteome Profiler Human Cytokine Array Kit, Panel A http://www.rndsystems.com/Products/ARY005. It only needs 0.5 mL cell culture supernatant to test 32 cytokine expression.

The suspension bead system (Luminex) is the only method that won’t ruin you. You can get premixed kit or custom made from several companies like those already mentioned (Millipore, BioRad, R&D Systems etc.).

Usually the kits comes with enough of bead allowing use of half or third amount numbers, so you can get more sample results from a single 96 well kit, Furthermore, as the well is not directly involved in the assay (is only a container) you can stretch the kit even more if you only use standard and controls on one of the plates (make new every time).

Most kit comes both as ordinary beads (requiring filter plates) or as paramagnetic beads (requiring a magnet).

If you stretch the kits you can get empty plates form several companies (we use Millipore).

If you have low concentrations in your samples, use the high sensitivity versions, but the usually is less sensitive compared to ELISA.

I have used BD CBA Flex Sets for cell culture supernantants and they worked well. Link below:

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

Definitely the Luminex xMAP platform, like Pal Bela Szecsi suggested. There are many distinct kits, flexibility of custom kits and the fact that you can assess several cytokines at once in each single well, allows you to reduce costs per analyte and to use only the amount of sample that you would typically use on a standard ELISA assay (where you would only assess one analyte). Also, the time an labor are reduced incomparison to the standard ELISA kits due to the multiplexibility.

Here's a useful link to find kits:

https://kitfinder.luminexcorp.com/?utm_source=cookbook+kit+finder+banner&utm_medium=banner&utm_content=kit+finder&utm_campaign=xmap+cookbook&__hssc=&__hstc&hsCtaTracking=fb90f630-729e-418b-8dad-7076125cd6bf|e5a7c553-4dfa-4799-b851-55c9f5b7d473

Melissa Vetten,

Were you able to get publishable results with the CBA method? 

thanks,

Elizabeth

Hi Elizabeth

Some of the cytokines I assayed fell below the level of detection for the kits, but I guess that is dependent on your cell line.

My results were reproducible, but we havent yet tried to publish the data.

Melissa

Some cytokine are at low levels unless you have som inflammation, but that is an answer too.

I have use the BD assay and published it:

The Open Breast Cancer Journal, 2012, 4, 11-17

but it was rather expensive to analyse.

Mulptyple cytokine assays based on Luminex technology! 

I have used and published with the BenderMed/Ebioscience Cytometric Bead Array.  I have used the kit many times with many different cytokines/chemokines.   I have found the dynamic range to be as good and the reproducibility to be even better than ELISA for MOST cytokines (you are getting essentially 100 Reads per sample per cytokine (low CV)) . What's great is you can use very little supernatant and detect at least 14 cytokines at once. The data is not expensive to analyze. It requires access and experience with a flow cytometer to be acquired but the data can be analyzed essentially like an ELISA utilizing a standard curve and  gMFI instead of your typical absorbance values.  If supernatant is scarce and you know your targets this works well.  Luminex is nice also but requires the proprietary software and machine.

Siebert JC, Inokuma M, Waid DM, Pennock ND, Vaitaitis GM, Disis ML, Dunne JF,

Wagner DH Jr, Maecker HT. An analytical workflow for investigating cytokine

profiles. Cytometry A. 2008 Apr;73(4):289-98. doi: 10.1002/cyto.a.20509. PubMed

PMID: 18163472.

Thank you so much for your sharing. In my lab, we just have Flow Cytometer instrument, so I think that I'll conduct experiment on CBA method (with limited number of cytokines). Hopefully, I can try this experiment on Luminex if my lab invest on a new Luminex system.

Thank you for your kind supports on my question.

Does anyone know laboratory or center in USA that can conduct Luminex experiment as a service (we can pay for this service)? I think that if my lab can't afford for a new Luminex system, we also can make it by service from other lab.

Thanks, Nathan,

I'll look up your publication. I had a poor experience with those beads with two different flow cytometers, two different sample types (one supernatant from incubated cells, the other frozen plasma). The standard curves were great, but standard deviation of samples was huge. Very few publications seem to use mutiple analyte bead-based flow cytometry still. I'm hoping the technology improves. 

We have had decent results with upto 20 cytokines/chemokines using Luminex based assays such as Milliplex.

we measured some with luminex and elisa in:

Bendsen, N. T., Stender, S., Szecsi, P. B., Pedersen, S. B., Basu, S., Hellgren, L. I., ... & Astrup, A. (2011). Effect of industrially produced trans fat on markers of systemic inflammation: evidence from a randomized trial in women. Journal of lipid research, 52(10), 1821-1828.

and othes with luminex:

Dantoft, T. M., Elberling, J., Brix, S., Szecsi, P. B., Vesterhauge, S., & Skovbjerg, S. (2014). An elevated pro-inflammatory cytokine profile in multiple chemical sensitivity. Psychoneuroendocrinology, 40, 140-150.

so it is possible.

how many samples do you have and what is you budget?

Cytometric bead array kits (cytometry) provide multiplex testing. Last kit we have used measured 18 cytokines in three tubes.

I agree with the Luminex recommendations.  I have used it to measure cytokines secreted into culture media and it worked very well.  I believe we used the 41 human cytokine/chemokine kit from Millipore a few years ago.  

Costs are high for luminex-based kits but it is possible to get custom kits if needed. 

I've successfully used a CBA-based assay (LEGEND-plex kit from Biolegend) for the simultaneous detection of 13 human cytokines from only 50ul of culture supernatant from in vitro activated human CD4 T cells. The assay was quite easy, and did not require plate vacuum washers and was easily run on a BD Canto flow cytometer. I used an ordinary 96 well plate to run the LEGEND-plex assay, rather than the plate provided in the kit (which is specifically designed for vacuum-based washing).  Entire assay including analysis took a full day, but well worth it compared to running multiple ELISA kits.