Question

I have a cell line (HeLa) that I want to grow in culture and then test the efficacy of a drug on the production of a protein identified to be antiproliferative (IkB-alpha). At this point, I have no idea as the the EC50 of the drug. Using your knowledge from the course and from the literature, describe in detail: How to grow the HeLa cells, including media and supplements, temperature, &as requirements. How to find the EC50 of the drug in terms of increasing expression of IkB-alpha (use the immunoblot to quantitative the amount of protein) how to determine where IkB-alpha is expressed in a cell culture using immunohistochemistry.

Answer 1

a. HeLa cells are adherent cells which attaches to the substratum of the cell culture flask. The media used for their growth is Dulbecco modified eagle medium (DMEM), which is supplemented with 10% Fetal bovine serum and 1% Pencillin/Sterptomycin antibiotic. The cells are grown at 37^oC with 5% CO₂.

b. Treat the HeLa cells in increasing concentration of drug for certain time period. Then isolate the total protein from cells and do a immunoblot with the antibody against the IkBa protein. If expression of this protein is half when compared to the control (Which is not treated with the drug), then you term this concentration as EC50.

c. Grow the cells on a slide and fix it with methanol to permeabilise the cell. Then add the required antibody against the protein and also a dye for visualising the nucleus. View the cell under a microscope to see co-localisation of the protein with cytoplasm or nucleus. If the protein co-localise with nuclear dye, then it is present in the nucleus. If does not co-localise with nuclear dye, then it is present in the cytoplasm.