Frequently Asked Questions

RealTime-Glo™ MT Cell Viability Assay

The complete protocol for this product is available in Technical Manual #TM431.

What type of multiwell plates should I use for this assay?

For optimum performance, use opaque, white multiwell plates that are compatible with your luminometer. Luminescence signal is diminished in black plates and increased well-to-well cross talk is observed in clear plates. RLU values, such as those shown in the figures of the Technical Bulletin, will vary depending on which plates and luminometers are used to generate data. Although relative luminescence output will vary with different instruments, this variation should not affect assay performance. Any solid white plates that fit your luminometer should be suitable. We don’t have specific recommendations but here are some examples:

Corning® Costar plate (Cat.# 3917)
Greiner Bio-One CELLSTAR plate (Cat.# 655073)
Nunc™ MicroWell™ 96-Well, Nunclon Delta-Treated (Cat.# 136101/02)
Falcon® 96-well Solid White Flat Bottom TC-treated (Cat.# 353296)

Are there any key considerations before starting the assay?

Determination of the assay linearity with a given cell type is imperative, especially with 3D cultures. Consider if the signal decreased because viability decreased, or simply because the substrate ran out. Review the section on determining assay linearity in Technical Manual #TM431.

Are there any considerations when preparing the reagent?

Initial solubility can be increased by using a lower concentration and by warming the RealTime-Glo™ reagent and cell culture medium to 37°C and mixing until the reagent is clear. If precipitates/particles are observed after addition of the reagent, they are not due to contamination, but rather substrate that is not fully solubilized or reduction products that accumulate over time. See the section on reagent precipitate removal in Technical Manual #TM431.

Will this assay work with cells that display high metabolic activity?

Cells with rapid doubling time, increased metabolism, and high numbers of cells will use the substrate more quickly. These factors could reduce how long the signal remains linear.

Is this assay compatible with 3D cell cultures?

Yes, the RealTime-Glo™ Assay has been tested in Matrigel™-based, collagen-based, and hanging-drop 3D spheroid cell culture models.

I don’t have an environmentally controlled plate reader. How can I get kinetic readings?

The following video explains how you can perform kinetic measurements without an environmentally controlled plate reader.

What instrument settings should I use for this assay?

Refer to the instrument manual for specifics. In general, most instruments have a standard luminescence setting that should collect all wavelengths available and have no filters. An integration time (time to collect the signal) of 0.25–1 second per well is a good starting point.