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    di Bernardo Lab - Systems and Synthetic Biology Lab
    You are here: Home Internal Site WET LAB CellTiter-Glo Luminescent Cell Viability Assay Protocol

    CellTiter-Glo Luminescent Cell Viability Assay Protocol

    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. 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

    1. Take the 96-well plate out of the incubator and equilibrate the plate and it’s content at room temperature for approximately 5 minutes.
    2. Discard the culture media from every well and replace it with 50 μl of PBS after a fast wash in PBS.
    3. Add 50 μl of CellTiter-Glo. to each well.
    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.
    6. Transfer the content from each well of the 96-well plate to a new opaque-walled multiwell plates
    7. Prepare control wells containing PBS without cells and CellTiter-Glo. to obtain a value for background luminescence.
    8. 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.