We have developed a rapid, sensitive, and inexpensive method for measuring the cellular
protein content of adherent and suspension cultures in 96-well microtiter plates.
The method is suitable for ordinary laboratory purposes and for very large-scale applications,
such as the National Cancer Institute's disease-oriented in vitro anticancer-drug
discovery screen, which requires the use of several million culture wells per year.
Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol)
sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four
washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered
Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density
in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were
linear with the number of cells and with values for cellular protein measured by both
the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered
supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000
cells per well. The sensitivity of the SRB assay compared favorably with sensitivities
of several fluorescence assays and was superior to those of both the Lowry and Bradford
assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric
end point that is nondestructive, indefinitely stable, and visible to the naked eye.
It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating
clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces
strongly with laser excitation at 488 nm and can be measured quantitatively at the
single-cell level by static fluorescence cytometry.