What are the steps protocol for Preparation of heat-killed P. acnes and the measurement of cytokine production using ELISA KIT ?
According to many publication protocol I need to seed 1X10^6 cells/well in 24 well cell culture plate.
And Stimulated with heat-killed P. acnes (wet weight 100 lg/ml) alone or in combination
with different concentrations
How can I calculate the volume of medium (containing cells) need to be taken to seed 1X10^6 cells per well? And for all wells what will be the total volume of medium required to seed the said amount of cell?
What the proper volume should I use to seed each well in 24 plate? Is it I ml
And volume for heat-killed P. acnes
With different concentrations of tested sample to added to each well?
You have to prepare the heat killed antigen by boiling the P. acnes for 5-10 mins and sonicate to make soluble antigen. Measures the protein contain. Then seed 1X10^6 cells/well in 24 well cell culture plate in 1 ml medium. Stimulate with heat killed antigen (you have to standardized the procedure with different concentration of antigen). You can use a positive control as PHA or LPS. After proper incubation, collect the supernatant and measure required cytokines by ELISA.
thanks,
In the Stimulation the cell with heated killed bacteria (100ug/ml) , the the bacteria suspension should be re-suspended in cell culture media or pbs buffer immediately before stimulation assays?
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