I want to evaluate the effect of a medication on a EAE mouse model and I defined the control positive,control negative and treatment group. If i want to separate Spleen mononuclear cells and culture it to calculate the Stimulation index. How will be Protocol?
Hi Hamidreza. It will depend upon what measure of stimulation you are going to measure. If you are going to calculate proliferation index, you first need to label your cells using CFSE or efluor dyes and then put in culture for 3-5 days. Cells treated with PHA are recommended positive control in this. Depending on the number of cell divisions, intensity of CFSE fluorescence signal after termination of experiment, will vary between the cells in the culture. Then you will need to calculate proliferation index as number of cells in all generations divided by number of original parent cells. Then proliferation indices can be comapred between your study groups. If you are going to detect any other marker as a measure of stimulation, then stimulation index will be the value of parameter obtained in your test divided by the value obtained in experimental control, like we do in real-time PCR.
I hope it will be of use.
Good luck
Hi Hamidreza,
According to the article of Zhou et al (2014) [DOI: 10.4103/2321-3868.126088], stimulation index (SI) = OD of responder cells in cells with stimulator cells added/OD of the same responder cells in wells containing the responder cells only. They used CCK-8 cell counting kit and measure absorbance at 450nm after 4 hours of incubation.
You may still find their calculation useful whatever proliferation assay method you would use. Good luck.
Sincerely,
JP
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