Dear Syed

in my experience, i work in mouse bacteria infection and i used the liver to collect the cytokines. i performed tissue lysate methods.

1. dissect the interest sample

2. used extraction buffer (you can use the commercial ones or make it by yourself)

extraction buffer contain lysis buffer (i.e. SDS and Triton) and protease inhibitor (I suggest you to use cocktail like Halts Protease inhibitor by Thermo)

3. agitation for 2 hr in 4oC

4. collect the supernatant by centrifuge in 14.000 rpm for 30 minutes

Good luck for your research

Hey Syed,

I performed ELISAs on liver and adipose tissue homogenates and they work well. You can homogenise the tissue with a non-denaturing lysis buffer, here is the link from Cell Signaling Technology Lysis Buffer which worked perfectly fine for me:

https://en.cellsignal.de/products/buffers-dyes/cell-lysis-buffer-10x/9803

This buffer contains triton, which is better compared to SDS as triton is a nonionic detergent. Make sure you add protease and phosphatase inhibitors as also suggested by Mawar. You want to minimise the possibility of denaturing your proteins if you want to perform an ELISA.

Homogenise the tissue in 1x cell lysis buffer (bead based homogenisation works great, you can also sonicate the homogenate for a few minutes - works even better then), I use 500 uL lysis buffer for 250 mg liver tissue but 1 mL for 250-300 mg adipose tissue, you can optimise this for brain. Important part is that you reconstitute &/ dilute your ELISA standards in the same buffer you homogenised your tissue samples with. I compared the regular standards from my ELISA kit with standards I reconstituted in the lysis buffer and I observed differences in the absorbances, so your standards have to be comparable to what you want to measure your unknowns from. After the homogenisation and sonication, centrifuge your samples to get rid of debris - for adipose tissue I do 10,000g for 10 min and I observe the lipid-fat layer on top and cell debris as the pellet, I then collect the internatant containing the proteins and perform BCA protein assay on it to see how much protein I have collected. You can again optimise the centrifugation step for brain. Another very important factor is that you measure your total protein levels (at least duplicates for this) and either dilute all your samples to the same protein concentration such that you add equal amounts of protein for each sample to your ELISA or that you normalise your ELISA results to total protein levels afterwards. I choose the normalisation to total protein afterwards which is much easier and works better if you have some samples with less protein recovery. Make sure you run a trial first and see whether you will need to dilute your homogenates at all or not. If they all end up too concentrated exceeding your highest standard you would waste a lot of time, effort and samples...

As for your flow cytometry protocol and combining purposes: as you homogenise the tissue the way you do it, before passing it through the filter you can collect some homogenate and use this for ELISA before diluting your proteins. I believe you want to keep your cells as intact as possible for flow cytometry, so you can further process the part you collect from your first homogenate as explained above.

Alternatively you can also stain your samples for intracellular proteins -both cytoplasmic and nuclear to detect cytokines, secreted proteins etc. at a single-cell level - and perform flow cytometry on them simultaneously. Here is a useful link for this with the kit suggestions http://tools.thermofisher.com/content/sfs/manuals/staining-intracellular-antigens-for-flow-cytometry.pdf

Another method would be western blot for which you would use RIPA buffer instead of non denaturing cell lysis buffer to homogenise your samples but this is only semi-quantitative and you would have to pool samples to fit them onto the gel... Just an idea if nothing works out but I am sure you will be fine with the ELISA. Let me know if you have any troubles/questions. Hope this helps.

Good luck!