Hello everyone,
After exposing HeLa cells to phthalate, I will detect various antioxidant levels with an ELISA kit. Before ELISA, I will determine the protein concentration of the cells with BCA assay. During BCA, after lysing my cells with RIPA buffer, I will dilute them 2x, 5x and 10x with distilled water. My question is, what is the number of cells that need to be seeded per well for BCA assay? I'm open to your suggestions. Thank you!
Hello Naile Merve Guven,
It would not be proper to give you the cell number because it will vary with cell type. So, you will have to perform a pilot experiment to decide the optimum cell density for a multi- well plate format which you will be using for the actual experiment.
In the pilot experiment, you may seed different cell densities and choose the optimum cell density that gives you 80% confluency in 24 hours. For instance, for HeLa cells in a 96-well plate format you may seed cells in the range of 5000 to 25000 cells per well in 200ul, and choose that cell density which gives you 80% confluency after 24 hours.
After having obtained the optimum seeding density from the pilot experiment, you may seed cells at that optimum cell density in whichever plate format you decide, and after 24 hours you may carry out the treatment.
After treatment, you may lyse the cells with RIPA buffer and determine the total protein of the lysate using BCA assay with different dilutions of the lysate.
Best.
- Badges
- Science method