Hello Taeyoon Jung,

After 24 hours, when treating the cells, you should use 10% FBS because the cells have to be healthy when you treat them. If cells are not provided with enough nutrition (for example, if cells are incubated with the drug for 72 hours in media without FBS) they may undergo apoptosis, and this in turn may affect your end result. There is a likelihood that you may interpret your results wrongly.

Also, the use of phenol red in growth media will depend on the type of cell viability assay you perform. For instance,

in MTT assay, phenol red may be a problem because the absorption spectra of formazan and phenol red overlap, and so you may get an added effect during the formazan absorption reading.

On the other hand, in Alamar Blue assay, there is no interference of phenol red in growth media. The presence of phenol red will just shift the values by a marginal unit higher.

The best way out would be to have sample background controls. For example, you may use well having only growth media without cells containing FBS and phenol red as blank to eliminate the background in the assay.

Best.