CellTiter-Glo Luminescent Cell Viability Assay Protocol
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. Buffer 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.
Protocol for the Cell Viability Assay
- Take the 96-well plate out of the incubator and equilibrate the plate and it’s content at room temperature for approximately 5 minutes.
- Discard the culture media from every well and replace it with 50 μl of PBS after a fast wash in PBS.
- Add 50 μl of CellTiter-Glo. to each well.
- Mix contents for 2 minutes on an orbital shaker to induce cell lysis.
- Allow the plate to incubate at room temperature for 10 minutes to stabilize luminescent signal.
- Transfer the content from each well of the 96-well plate to a new opaque-walled multiwell plates
- Prepare control wells containing PBS without cells and CellTiter-Glo. to obtain a value for background luminescence.
- Record luminescence.
Note: Uneven luminescent signal within standard plates can be caused by temperature gradients, uneven seeding of cells or edge effects in multiwell plates.
Note: Instrument settings depend on the manufacturer. An integration time of 0.25–1 second per well should serve as a guideline.