I would like to take the organ from the mouse and analyze the cytokine levels.
I would do an ELISA. What do you think? Are there other possibilities to solve the problem?
What should I use as a reference? GAPDH or amount of cells?
Thanks Rob.
Thats a good idea.
And I will just smash the organ in PBS, centrifuge it (300g) and take the supernatant for the analyzation.
best,
Sebastian
If you are measuring mRNA then GAPDH makes sense. for cellular studies, you isolate the lymphocytes from spleen. culture them overnight and measure the cytokine secretion in the culture media using luminex bead array. you can normalize the data to number of cells.
You will not be able to measure the cytokines by the method proposed by you.
What are you looking for? When people are looking for immune response in spleen, they will often make a single cell suspension and look for ability of T cells to produce cytokines upon re-stimulation either by flow cytometry (intracellular cytokine) or by ELISpot. While this does not tell you what cytokines are present in the spleen and to what amounts they are being produced, it will tell you the potential of cells to make specific cytokines.
I would like to treat the mice and take then the organ.
That means, I would like not to put the cells in culture -> side effects from MACS and isolation (too much stress)
is there another way and not to put the cells in culture?
in all immunological studies spleenocytes are isolated with MACS. a proper control will take care of the side effects. or you can do real time RT-PCR which does not represent the real scenario. other thing you can do a intra cellular staining and read on FACS
Thanks!
But why do you think it would not work out to smash the organ in PBS - centrifuge - take the supernatant and analyze that ?
I know it might be difficult but I think it could work.
I think it would be interesting to put spleen cells in culture and to do a sample unstimulated and another one stimulated (with plate bound CD3 or peptide if you did antigen specific stimulation or allogeneic cells if you did allotransplant). normally the spontaneously secreted cytokines are very low - this is why you would rather analyse the response after restimulation. You could also use PMA + Ionomycin + Brefeldin A to look by intracellular antibody staining
thanks a lot!!
My experiment would now look like that:
stimulate the mice
perfuse the organ
smash the organ
centrifuge and take supernatant for analysis
parallel I will also use unstimulated mice as a control
in addition I could isolate the cells of the organ of stimulated and unstimulated mice, bring it into cell-culture and take the supernatant after 24hours
best,
Sebastian
Hi there,
I'm just wondering how you will account for the hemolysis of red blood cells in the spleen, wouldn't the hemoglobin effect the ELISA readings?
Thanks,
Anna
Hi,
I used a red blood lysis buffer...
all the best,
Sebastian
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