Hi, there is some details that will help to understand your system, the first thing would be: what kind of cells and time-points are you using?

Hi Noman, is there any reason why you have chosen to use Western Blot? Do you have samples for use with ELISAs or similar methods? Or perhaps you could look at the mRNA levels. Perhaps these cytokines are expressed at very low levels or it may be that you have missed the time point at which they are expressed optimally and, therefore, you may be better off looking at mRNA levels.

for multiple cytokines paople are using Luminex or MSD technology

Go bead assay by flow cytometery.

I agree with Pedro and Jose. To my knowledge, one can figure out clear cytokine profiles. For example: in my Mono-Mac-6 assay, IL-6 comes later than TNF-α, when stimulated with living bateria. So, I think, there are two opprtunities: either you have no IL-1β/IL-6 expression or you just miss them...performing time kinetics is always useful.

Thank you every one for your prompt and Valuable reply.

Pedro Escoll: we are using THP1 and HUVEC cells and Time periods are 4,8,12,24 and 48 hrs.

Jose Ruffino: we got result through mRNA. Westrn blot is to confirm the result and response at different time points.

Kalyan Manda: yes your suggestion is valuable, i wll try immunofluresent staining.

Bead assay and Luminex not avalible or new for our lab.

Dogan Demircioglu: previous litratures and mRNA given result but not Westrn blot. :(

Areej Assaf : yes after reading some more litrature i wll try ELISA. and with different time points.

You can also use flow cytometer for detection of cytokines ex. test CBA method (BD-Bioscience) works on the simplest 3-4 colours flow cytometer. This type of tests give you results for 6 cytokines from 50 ul of volume. You can create the standard curve and have pg/ml value.

Hi Noman, you are right, as cytokines are secreted after stimulation is better to look at the supernatants and not inside the cells. Try with ELISA as you said and the time-course you are already using (4, 8, 12, 24, 48h). You should have nice results with THP-1 cells, but I'm not sure with HUVEC as I never worked with them and perhaps are not secreting these cytokines. Remember to differenciate THP-1 cells to macrophages adding PMA (50ng/ml during 3 days) and be careful of not taking cells while harvesting supernatants (don't touch the bottom of the well and if you are not sure, centrifuge the supernatants and discard pellets). These detached cells in the supernatants could mask your results as the cytokines could come from inside the cells and not from the supernatants, functional relevance of the cytokines is as secreted proteins. Good luck!

You can measure cytokines in supernatant and in the cell lysates - depending of the material You have. I agree that most cytokines are secreted to the supernatant.

Hi Noman. You have some good suggestions already, and actually in my lab we use multiplex assay for human cytokines and detection by flow cytometry. The kinetics are very important, but for sure you should see IL1b secreted from THP1with a peak (about 3,000 pg/ml) at 8 h. IL6 is poorly secreted by THP1, the majority of IL6 remains intracellular. We have no experience with HUVEC. In any case, you may check what your antibody to IL1b recognizes. It might be that the antibody recognizes a bio-active IL1b only, while the cytokines on your membrane are denatured after electrophoresis and transfer to the membrane. Hope this comment is helpful.

Hi Norman,

If you are not seeing pro-IL-1B in blots of cell lysates, then try a different antibody - it really should be there in THP-1s. Some IL-1b Abs are great at detecting pro-IL-1b, but not the bioactive form. If you are looking in supernatants, try methanol/chloroform precipitation of proteins to clean up and concentrate the samples. You may need to reduce the amount of FCS/sera in your assays as well. Alternatively, you can use an IP-type method, using beads labelled with anti-IL-1b (or IL-6) to collect the cytokine from the supernatants, then run on a Western.

thank you for your advice.

James Harris: yes we are thinking on different troubleshooting in western blot. and also consider some other method as suggested.

Thank you once again.

The best would be ELISA. But if want to detect the mature form by WB, check the paper from Olaf Gross entitled "measuring the Inflammasome" there you have all the hints

i would like to know at what time hour it is good to take cell culture supernatant for cytokines estimation(Il-6, Il-1beta, TNF alpha)

also is there any need to condense the supernatant or what amount of culture media to be taken?

confusion is that cytokins are very minute amount so how to increase concentration in treated culture?

James different cells behave differently. its depends on what kind of cell line you are using. and i am not sure about your second question.

Its not ideal to rum western blots for cytokines. Why not using ELISA.

Although Immuno-fluorescence in tissues are other options

Are you dedicated to that particular IL1b antibody? In my experience (and in the literature) the AF-401-NA antibody for R&D is the best for western blot.