Standardized sulforhodamine b colorimetric cell proliferation assay for anticancer activity screening in educational...

The sulforhodamine B (SRB) assay is a robust and cost-effective methods for assessing cellular proliferation and cytotoxicity in vitro. Originally designed to support drug screening efforts at the National Cancer Institute, this colorimetric assay quantifies protein content as a proxy for cell number and has proven reliable across diverse cancer cell lines. We present a fully standardized, step-by-step SRB assay protocol optimized for high-throughput application, educational accessibility, and translational relevance. Our streamlined protocol utilizes a fixed seeding density and simplified workflow, allowing for reproducible results with minimal technical expertise or specialized equipment. It has been successfully integrated into undergraduate teaching laboratories, while also supporting preclinical drug discovery efforts, including the identification of nitrosylcobalamin (NO-Cbl) as a novel anticancer agent. Key features of this protocol:•Provides an easy-to-follow format for generating reproducible cytotoxicity data in 96-well plates.•Can be used by novice researchers in teaching or early-stage discovery labs.•Suitable for scaling to support high-throughput compound screening campaigns.This accessible and validated SRB protocol has the potential to accelerate cancer research and empower future scientists at the bench.