Typically, we recommend harvesting the culture medium (i.e. the supernatant) 24h post-transfection for the GLuc assay. However, depending on the type of promoter that drives the GLuc expression, the amount of GLuc-expressing vector used per transfection, the transfection efficiency and the health of the cells, the GLuc activity can be detected earlier than 24h post-transfection. For example, eight hours after transfecting HeLa with pCMV-GLuc (i.e. 80% cell density, 1µg pCMV-GLuc and 4µL of TransPass HeLa Transfection Reagent per well of a 12-well format), the GLuc activity was detectable in the culture medium.