CellTiter-Blue for use in viability assays

May 01, 2015

Rice University
BioEngineering
Researcher

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Company:

Promega

Product Name:

CellTiter-Blue

Catalog Number:

G8081

CellTiter-Blue was used to evaluate the viability/proliferation of U2OS cells (ATCC) treated with PLK1 or negative control siRNA to determine the appropriate positive control concentration to measure siRNA delivery/expression knockdown in future experiments.

Experimental Design and Results Summary

Application

Cell Viability

Starting Material

U2OS cells from ATCC

Protocol Overview

Reverse transfection of U2OS cells performed in 96-well plates with HiPerFect transfection reagent: siRNA diluted in OptiMEM media to desired concentrations and mixed with 0.5 uL HiPerFect reagent per 50 uL OptiMEM then added to 96-well plate and allowed to form complex for 10 minutes at room temperature. After incubation, 5000 cells/well were added in 100 uL complete media and swirled briefly, then incubated at 37'C in a 5% CO2 incubator. At 72 hours, cell viability was assessed by addition of 20 uL CellTiter-Blue/well and further incubation for 4 hours at 37'C. After incubation with viability reagent, fluorescent measurements were taken on a DTX 880 multimode detector using the preset CellTiter Blue protocol.

Tips

CellTiter Blue incubation time is highly dependent on cell line and protocol, recommend taking measurements hourly (or every 2 hours) until protocol is optimized for your purposes.

Results Summary

CellTiter Blue fluorescent readouts of PLK1 siRNA-treated U2OS cells were normalized to untreated cells and analyzed in Prism to determine optimum siRNA concentration for measuring efficiency of siRNA delivery/knockdown in future experiments. 20 nM of PLK1 siRNA was determined to be the optimal concentration for usage as a positive control in our cells with the described reverse transfection protocol.

Additional Notes

CellTiter Blue was tested as a proliferation/viability reagent when our normal MTS/WST-1 assays were not repeatable with siRNA experiments

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Summary

The Good

Precise, accurate results for measuring cell viability

The Bad

Some optimization required, requires fluorescent detection methods

The Bottom Line

CellTiter Blue has become a mainstay in our lab for evaluating cell proliferation and viability for experiments involving siRNA transfection.

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