I am doing cytokine ELISA using supernatnat collected from conditioned media of different treatment. However in my different treatment cell growth is very different, I mean cells grow very slow under treatment condition wrt to control condition. In that case how should I normalise my ELISA quantification readings.
Hi,
Once you collected your media, what do you do with the cells? You can collect the total proteins, quantify them and normalize the cytokine concentration with each corresponding total protein concentration. The total protein should be proportional of your cell number.
Dear.. you must have a control group that should be kept without any treatment and in a healthy condition to compare results over long experiments.
Thanks Christine for your answer. I have planned my experiment similarly, but I had a doubt whether I should quatify total protein in conditioned media of respective treatment and normalise to that or the first one will be more appropriate.
Hi Umesh,
To minimize the variation in your result you may count the number of cells following collection of conditioned media. Later on you may normalize the expression of paracrine factors per million of cells for a particular treatment.
Best wishes,
NH
Hi
If you counted the cell number when playing( for example 1 million cell per each well of 6 well plate)then you did the right thing. When you plan your experiment leave 2 well as controls.one have the media alone and the second have the cells with the media.
Wish you the best
Hi Umesh
At the beginning of the experiment, you have to start from one number of cells in the flasks or plates by using Neubauer Haemocytometry and put duplicates in the experiment without treatment (control), after the treatment and centrifuge; you will be able to compare the treated with nontreated (control) cells correctly.
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