Hi
I need to measure cytokines (with an ELISA kit) and nitrite (with Griess reagent) in cell media of BV-2 microglial cells.
According to the cytokine assay protocol i get the results in pg/ml. How do you compare the effect of different treatments? Do you calculate protein concentration and then divide the result of cytokine in this protein concentration?
The same question as to nitrite- i get results in micromollar. Do i need the protein concentration of the samples?
Thanks
Yes and No. It depends on what happens to your cells, when you treat them. When you take cell supernatants, they contain proteins from cell metabolism and from cells dying during that Experiment. If one treatment raises death rates, you will have a lot more protein in your medium, Independent from whether your cells produce more or less of the cytokine of interest. So first you have to look at cell-numbers, deathrates or apoptosis/necrosis. If these values are similar in different experiments, you can use protein concentration in your quotations, but be aware that cytokine-concentration measurements are quite exact, while cell counting and protein- measurements are not, so you'll need a lot of "n"s.
Thanks. I treat bv-2 microglial cells with LPS. It affects their cell death and also i have a lot of floating cells. So should i centrifuge my samples and then take supernatant for cytokine ELISA and the cells for protein concentration?
Thanks a lot
Hi,
I agree with Markuz. Actually you should care about necrosis, because cell content is released. Apoptosis it's a physiological process where cell content remains inside apoptotic vesicles, so this won't interfere in your cytokine determination.
Yes, I think you should centrifuge your samples. Take account that if these remaining cells break during the process, you'll be overestimating cytokine concentration.
Be aware that treatments also can modulate total protein concentration in supernatant.
Good luck!
For cytokines, ng or pg/ml is the expression used while the conditioned media is prepared using constant number of cells/ml, for constant duration.
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