Cell culture: the foundation under every assay number
Every number measured on cells inherits the history of the culture it came from. Three properties define that history.
Confluence is the percentage of the dish surface covered by cells. Most cell lines are passaged — split into fresh dishes — at 70–90% confluence, because beyond that point contact inhibition changes gene expression and nutrient depletion shifts metabolism. An assay seeded from an over-confluent flask will not behave like one seeded from an exponential-phase culture, no matter how careful the pipetting.
Passage number counts how many times a culture has been split. With each passage, the population drift: slow-growing variants take over, karyotypes change, and immortalised lines slowly stop resembling their original biology. Comparing low-passage and high-passage cells is comparing different experiments.
Viability — the fraction of living cells — is the third pillar. Dead cells release proteases and DNA that raise background in almost every assay, so viability (typically measured by trypan blue exclusion or a luminescent assay) should be checked whenever a result looks surprising.
Seeding density deserves the same respect. Cells behave differently at low density (poor conditioned medium, weak paracrine signalling) and at high density (nutrient stress, contact inhibition). Assay protocols specify a seeding number for a reason: it places the cells in the linear range where a measured change tracks a real biological change.