A. Reagent Preparation
1. Thaw the CellTiter-Glo® Buﬀer, and equilibrate to room temperature prior to use. For convenience the CellTiter-Glo® Buﬀer may be thawed and stored at room temperature for up to 48 hours prior to use.
2. Equilibrate the lyophilized CellTiter-Glo® Substrate to room temperature prior to use.
3. Transfer the appropriate volume (10ml for Cat.# G7570 and G7571, or 100ml for Cat.# G7572 and G7573) of CellTiter-Glo® Buﬀ er into the amber bottle containing CellTiter-Glo® Substrate to reconstitute the lyophilized enzyme/substrate mixture. This forms the CellTiter-Glo® Reagent.
4. Mix by gently vortexing, swirling or inverting the contents to obtain a homogeneous solution. The CellTiter-Glo® Substrate should go into solution easily in less than 1 minute.
B. Protocol for the Cell Viability Assay
1. Prepare opaque-walled multiwell plates with mammalian cells in culture medium, 200µl per well for 96-well plates or 50µl per well for 384-well plates. Multiwell plates must be compatible with the luminometer used.
2. Equilibrate the plate and its contents at room temperature for approximately 30 minutes.
3. Add a volume of CellTiter-Glo® Reagent equal to half the volume of cell culture medium present in each well (e.g., add 25µl of reagent to 50µl of medium containing cells for a 384-well plate).
4. Mix contents for 2 minutes on an orbital shaker to induce cell lysis.
5. Allow the plate to incubate at room temperature for 10 minutes to stabilize luminescent signal.
Note: Uneven luminescent signal within standard plates can be caused by temperature gradients, uneven seeding of cells or edge eﬀect in multiwell plates.
6. Record luminescence with a plate reader.