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For collecting the secretome, a serum-free medium is typically used. As serum-starvation is not well tolerated by several cell types, the length of incubation in serum-free medium should be precisely calibrated in order to avoid protein release due to cell death.
Conditioned medium preparation
Seed HCT116 cells in a 6-well multiwell plate (or in a 35mm plate) at a density of 1.2M cells/well and incubate cells at 37°C and 5% CO2 for 24-48h [proceed to the next step when cell visual confluency is around 80%-90%]
Discard medium and wash cells twice with PBS
Add 2 mL of serum-free (antibiotics-free, if desired) medium (i.e., DMEM or OPTIMEM) and incubate cells at 37°C and 5% CO2 for 6h [the incubation in serum-free medium may be shortened or extended based on the experimental settings]