Why does seeding density change the result of a cell-based assay?

Why

Start with the arithmetic. A well seeded sparsely has room to grow through many divisions, while a dense well reaches confluence and stops. Because endpoint viability assays compare treated with untreated, and the untreated control has grown differently, the ratio changes.

Hafner and colleagues showed exactly this dependency on division number, which is why growth rate metrics were proposed. Then the chemistry. A fixed amount of compound shared among more cells means less per cell, and compounds that are taken up or metabolized can be depleted.

Medium is consumed faster at high density, so nutrient limitation and acidification appear. Finally the biology. Confluent cells contact-inhibit, change their cycle distribution and alter signaling, which matters for pathways sensitive to adhesion or to serum.

In practice, choose a density so that untreated cells are still growing exponentially at the endpoint, keep it constant across comparisons, record it, and verify it rather than assuming the dispensing was accurate. When comparing cell lines that grow at different rates, matching density at the end is not the same as matching it at the start, and the metric must handle that.