Section8:Cell Culture and Treatments

From Assay Guidance Wiki

Culture conditions can be adapted to any cell type. The following recommendations are based on our collective experiences with primary cells, normal and tumor cell lines, attached and suspension cells. Validated multi-channel pipettors can be used throughout. For experiments involving more than 8 to 10 plates, a multi-drop is useful.

1. Seed cells in a 96-well plate at near confluence 1 day prior to the experiment in 100 ul of growth medium. Optimal seeding density should be experimentally determined (see below).
2. For poorly adherent cells, it is useful to plate cells in wells coated with poly-D-lysine or other extracellular matrix components.
3. To avoid chemiluminescent signal spillover between wells, cells should be plated on white plates. Clear bottoms are convenient because cells can still be viewed under the microscope. However, there is a tendency towards “edge effects” with these plates. Opaque bottom white plates can minimize this problem. Alternatively, outside rows and columns can be excluded from the experimental format. When a fluorescent readout is being used a black plate should be used.
4. For growth factor-stimulated responses, the signal window is often increased if cells are serum starved prior to the experiment. Starvation can be for 2-4 h or overnight depending on the cell type and should be optimized using the experimental design templates (see example below).
5. Preincubate with compounds ∼1-2hr before adding the activating stimulus. Compounds should be added such that the final DMSO concentration is ≤0.25%. The compounds are diluted into medium containing serum or if in serum free conditions add BSA to a final concentration of 1%. Dilute the stocks serially to a final concentration of 10X. You will be adding 10 ul to a total of 100 ul to give a final concentration of the compound of 1X.
6. Prepare activators as 10x stocks in medium + 1.0% BSA. This is usually enough BSA to prevent peptides and small molecules from sticking to the sides of the test tubes. Return cells to incubator for the appropriate time.
7. Stimulate the cells with the reagent that is known to specifically produce the desired response. The concentration of the stimulant and treatment times will be determined by experimental design.