I am trying to quantify the cytokine levels using ELISA at different time points such as 12h, 24, 48 & 72h, from mouse endothelial cells upon hormone treatment. The question is regarding the cytokine release correlated to the cell numbers, since at higher time point there would be a higher cell number present. So, how to normalize the cytokine levels to ascertain that the increase in their level is due to time of treatment and not the cell number?
I would measure concentration of a total cellular protein in cell lysate from culture wells and normalize the cytokine concentration to cytokine amount/ cellular protein, e.g. 30 ng/ug cellular protein. Alternative way is a quantitation of copy number of genomic DNA by real time PCR, and then normalize your data per cells.
I was just curious to plot a cytokine release curve with respect to e time points. For independent time points, comparison with the untreated control surely holds valid. Thank you for the answer!
I would measure concentration of a total cellular protein in cell lysate from culture wells and normalize the cytokine concentration to cytokine amount/ cellular protein, e.g. 30 ng/ug cellular protein. Alternative way is a quantitation of copy number of genomic DNA by real time PCR, and then normalize your data per cells.
We are doing our tests as Tomozumi suggested to do, namely normalize our data to the total protein contents in each well. For us it is working very well even in very discrete tests as MDR activity quantifyed by fluorescence. Another tip - use more than triplicates in each test because you will see that for adherent cells there are growth differences from one well to the other even being very precausious when you seeded the cells.
In my lab we had some experience over expressing VEGF in adipose-derived cells, which started to proliferate after infection. So we plainly normalised by simple Trypsin detachment of cells and counting them with Trypan Blue stain. Then we used ELISA for VEGF concentration measurement and then normalised it to a fixed amount of cells, like 10^5 cells, or any you choose. It helped us. Good luck!
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA