The protocol is for time dependent study for screening of anti-HBV activity by analyzing of HBsAg and HBeAg expression (HepG2.2.15, 96 well plate)?
1- Time course (duration of study) to measure the efficacy activity of my candidate drugs
2-How long can i keep the drugs with cells in 96 well plate ? Should i change the media containing drug dilutions every 2 days?
3- What is the optimum hepg2.2.15 cell2 concentration range/ ml,? and the volume/ml?
4. With time cells grown in wells be come 100% confluent >>over growth>> cell death. How to manage this ?
5. in some published article, hepg2.2.15 cells are grown in serum free media, WHY??
I think there should be 2 approaches before u test ur candidate drug/molecule.
First to check the growth pattern of ur cells to know the exact time for subculture applying metabolic viability assessment through MTT dye conversion-this is related to confluency.
secondly,evaluate the expression pattern of HBsAg/HBeAg in ur metabolically viable confluent cells and select the linear range of expression.
Now u can apply ur drug in both ways i.e. start prior to introduce HBV and in parallel after the expression of antigen starts.Off course u can also try with different concentrations for fixed duration and fixed concentration for differen durations based on ur previous standardisation.
Hope this will help u.
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