Standardized sulforhodamine b colorimetric cell proliferation assay for anticancer activity screening in educational and research laboratories

Abstract

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.

Graphical abstract

Specifications table

Subject area:	Pharmacology, Toxicology and Pharmaceutical Science
More specific subject area:	Cancer drug screening
Name of your method:	Sulforhodamine B (SRB) colorimetric cell proliferation assay
Name and reference of original method:	Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990 Jul 4;82(13):1107-12. doi:10.1093/jnci/82.13.1107. PMID: 2359,136.
Resource availability:	https://dtp.cancer.gov/discovery_development/nci-60/

Background

Despite decades of scientific advancement, cancer remains one of the most complex and devastating diseases worldwide [1]. A critical barrier to progress lies in the early stages of drug discovery specifically, the identification and validation of compounds with antiproliferative effects [2]. While high-throughput screening (HTS) platforms exist in academic and industry settings, they are often cost-prohibitive, technically demanding, and inaccessible to most undergraduate and high school laboratories [3]. Yet, it is precisely in these grassroots educational settings where the seeds of future cancer breakthroughs can be sown (Table 1, Table 2, Table 3, Table 4).

Table 1.

Evolution of the sulforhodamine B (SRB) assay: from validation to high-throughput translation.

Parameter	Original SRB protocol (Skehan et al., 1990)	NCI-60 methodology (DTP/NCI Program)	Revised protocol (this work)
Initial Cell Seeding	Based on doubling time	Individually calculated per cell line	Fixed at 2000 cells/well for all lines
Confluency for Endpoint	∼100 % at endpoint	Variable, based on growth curve	Standardized at 90 % (96 hrs for untreated)
Purpose	Assay validation	High-throughput anticancer screening	Translational research & undergraduate training
Throughput	Medium	High	High with simplified, scalable workflow

Table 2.

Estimated time for each step in the SRB assay.

Step	Time required
Cell seeding	∼2 h
Drug dosing	∼1 h
Incubation (treatment)	72–96 h
Fixation and SRB staining	4–5 h (with drying)
Plate reading & analysis	∼2 h

Table 3.

Troubleshooting guide for the SRB assay.

Problem	Possible cause	Solution
High background in wells	Incomplete washing	Repeat acetic acid wash steps
Low signal	Low cell seeding or loss during wash	Increase cell number or pipet gently
Poor adherence post-seeding	Plate not equilibrated or low FBS	Use tissue culture-treated plates with 10 % FBS

Table 4.

Recommended media types for cell lines used in SRB assay.

Media type	Cell lines
RPMI	Jurkat, Molt 4, HUT 78, U266, U937, NIH—OVCAR-3, WM9, LnCAP
DMEM	MCF-7, ACHN, DU145, A549, KU-2, A375, HFF
DMEM-F12	BAEC

To address this gap, we have optimized and standardized a methodology based on the sulforhodamine B (SRB) assay for evaluating compound-induced cytotoxicity. Originally developed by Skehan et al. (1990) [4] and widely adopted by the National Cancer Institute’s Developmental Therapeutics Program (DTP) [5], the SRB proliferation assay offers a cost-effective, reproducible, and scalable alternative to enzymatic or metabolic-based assays [[6], [7], [8], [9], [10], [11], [12], [13]]. Multiple refinements have since emerged, including improved fixation (Papazisis et al., 1997) [14], reduced TCA toxicity (Vichai & Kirtikara, 2006 [15]), and comparative validations against neutral red and MTT assays [16]. The SRB assay was successful in the discovery of several compounds that are currently used in cancer medicine [17,18]. Our laboratory built upon these foundations to create a reproducible, undergraduate-accessible assay that enables standardized screening across cancer types.

Our adaptation simplifies the workflow by using a fixed seeding density (2000 cells/well), multichannel pipetting steps, and a clear, logical sequence that eliminates the need for advanced instrumentation or training. Most importantly, the protocol maintains high fidelity with established methods, enabling its integration into both teaching labs and translational research pipelines (Fig. 1, Fig. 2, Fig. 3).

Fig. 1.

Reproducibility of NO—Cbl Cytotoxicity: SRB assay (Cleveland Clinic (CCF vs NCI60). This figure highlights the concordance between two independent datasets—one from the JNCI publication and the other from the NCI60 screen—both utilizing the SRB colorimetric assay to assess NO—Cbl cytotoxicity. Despite being generated by separate organizations, the ID₅₀ profiles across cancer types show strikingly similar trends, with hematologic malignancies and ovarian cancers demonstrating greater sensitivity, while normal cell lines, including fibroblasts and endothelial cells, exhibit higher resistance.

Fig. 2.

Statistical comparison between NO—Cbl cytotoxicity data and NCI60 screening data. Pearson correlation analysis showed a strong positive correlation (r = 0.94), though not statistically significant (p = 0.23) due to a small sample size (n = 3). Spearman rank correlation revealed a moderate directional trend (ρ = 0.50, p = 0.67). One-way ANOVA demonstrated no significant difference in means across groups (F = 0.00027, p = 0.99), indicating strong agreement between datasets.

Fig. 3.

Step-by-step workflow of the standardized sulforhodamine B (SRB) assay. The figure illustrates the main stages of the assay: (1) cell plating (2000 cells/well), (2) drug treatment with 72–96 h incubation, (3) fixation using trichloroacetic acid (TCA) refrigerated for ≥1 hour, (4) washing and drying to remove unbound dye and debris, (5) staining with SRB dye for 30 min, and (6) solubilization and absorbance reading to quantify cell density.

The motivation for sharing this protocol is rooted in both scientific necessity and educational empowerment. At Walsh University, this method has been deployed successfully in the undergraduate curriculum, giving students authentic exposure to cell culture, pharmacology, and data analysis [19,20]. Its robustness and simplicity have also allowed high school students to engage in cancer research projects under guided mentorship, fostering scientific curiosity and competency at an early stage. These settings not only build laboratory proficiency but also instil a sense of agency in solving global health problems.

Our own work with nitrosylcobalamin (NO—Cbl), an investigational anticancer compound, underscores the power of this method [[21], [22], [23], [24], [25], [26]]. The SRB assay enabled us to observe reproducible dose-dependent cytotoxicity across multiple cancer cell lines which led to further validation through the NCI-60 screen and a pre-IND submission to the U.S. FDA. But this progress began not in a large pharmaceutical lab, but at the bench, one 96 well plate at a time.

We believe that the more hands we have at the bench screening compounds against cancer, the greater our collective potential to identify new therapeutic candidates. It is in this spirit that we offer this streamlined SRB assay protocol as a foundational, accessible tool that bridges education and discovery, with the hope that it will foster both.

Innovation

Unlike metabolic-based assays (such as MTT, XTT, and resazurin), the SRB assay quantifies total cellular biomass by binding stoichiometrically to basic amino acid residues in cellular proteins via charge interactions, making the readout independent of metabolic state or mitochondrial activity. This feature is particularly advantageous for screening agents that may directly or indirectly alter cellular metabolism, as the SRB assay provides a true measure of cell mass rather than relying on enzyme activity or redox potential. The technique’s sensitivity allows for the detection of as few as 50 cells per well, and the colorimetric endpoint is both stable and visually inspectable.

Another unique advantage of the SRB assay is that adherent cells do not require enzymatic detachment or trypsinization prior to analysis. Instead, cells are directly fixed in situ within the wells using trichloroacetic acid, thereby preserving monolayer integrity and minimizing cell loss or experimental variability before further manipulation. This fixation step streamlines the workflow, reduces handling artifacts, and further enhances the reproducibility and scalability of the assay for high-throughput applications.

While the SRB assay is already well-established for high-throughput cytotoxicity screening in standard 2D cell culture, future innovations may further expand its application. Potential developments include adaptation to 3D cell culture [27] and patient-derived organoid models, and possible integration with multiplexed assays.

Method details

Materials and methods

• •96-well flat-bottom tissue culture plates
• •Hemocytometer
• •multichannel pipettes
• •DMEM, RPMI-1640, or appropriate media (CellGro/Mediatech)
• •FBS, GlutaGro, antibiotic/antimycotic, and Plasmocin
• •Trypsin/EDTA
• •10 % Trichloroacetic Acid (TCA)
• •SRB dye (0.4 % w/v in 1 % acetic acid)
• •1 % Acetic acid
• •10 mM TRIS base (pH 10.5)
• •Plate reader with absorbance set at 570 nm
• •Sterile 15 and 50 mL conical tubes
• •Cell lines: MCF-7, Jurkat, A549, DU145, etc.

Standardized SRB assay protocol

All procedures described herein were conducted in accordance with institutional biosafety and laboratory protocols.

Procedure

 NOTE: All supplies must be sterile and tissue culture-grade. Label only the bottom of the plate (not the lid), as lids can be easily interchanged, leading to data errors.

Initial Cell Recovery and Expansion

• 1.Remove cryovial of frozen cells from liquid nitrogen or −80 °C freezer using appropriate PPE and handle with forceps.
• 2.Immediately thaw the vial in a 37 °C water bath for approximately 2 min or until a small ice chunk remains.
• 3.Transfer contents of the vial to a sterile 15 mL conical tube containing 10 mL of pre-warmed complete media.
• 4.Centrifuge at 150 × g for 5 min at room temperature. Carefully aspirate the supernatant to remove DMSO.
• 5.Resuspend the pellet in 10 mL of appropriate complete media and transfer to a tissue culture-treated 10 cm dish.
• 6.Incubate the dish at 37 °C with 5 % CO₂. Monitor cell adherence and morphology daily.
• 7.Once the culture reaches ≥90 % confluency, proceed with the assay protocol.

Assay procedure

• 1. Cell Harvest and Suspension.

•  NOTE: All supplies must be sterile and tissue culture-grade. Label only the bottom of the plate (not the lid), as lids can be easily interchanged, leading to data errors.

• 1.1 Turn on the centrifuge and set it to 150 × g (≈1000 rpm depending on rotor); allow it to pre-cool to 4 °C.

• 1.2 Aspirate media from a confluent 10 cm cell culture plate using a sterile pipette.

• 1.3 Wash the cell monolayer with 2 mL of 1X sterile PBS (e.g., Mediatech #21–040-CV). Incubate for 1 min.

• 1.4 Aspirate PBS completely, then add 4 mL of 1X Trypsin/EDTA (e.g., Mediatech #25–053-CI). Incubate for 5 min at 37 °C.

• 1.5 Neutralize trypsin with 10 mL of complete media (DMEM or RPMI + 10 % FBS + 1 % antibiotic/antimycotic + GlutaGro + Plasmocin).

• 1.6 Pipette up and down to ensure detachment, then transfer to a sterile 50 mL conical tube.

• 1.7 Centrifuge for 5 min at 150 × g (room temperature).

CRITICAL STEP: Mark the tube side facing the rotor to track pellet location. Always balance the centrifuge.

• 1.8 Carefully aspirate supernatant without disturbing the cell pellet using a sterile Pasteur pipette.

• 1.9 Resuspend the pellet in exactly 10 mL of complete media. Label as 'Stock'.

• 2. Cell Counting and Dilution

• 2.1 Transfer 150 µL from the 'Stock' to a new conical tube.

• 2.2 Add 10 µL to a hemocytometer using an L10 pipette.

• 2.3 Count 5 squares under the microscope and calculate the average cell count.

• 2.4 Multiply average by 10⁴ to determine cells/mL.

• 2.5 Enter values into Excel template:

• • Volume/well: 200 µL

• • Cells/well: 2000

• • Wells: 150

•  NOTE: Our protocol uses a fixed seeding density rather than varying it by doubling time.

• 2.6 Calculate plating volume (e.g., 2.5 mL cells + 27.5 mL media).

 Helpful Hint: Save your Excel file under a new name with today’s date. Do not overwrite templates.

• 3. Plate Setup

• 3.1 In a sterile 50 mL conical tube labeled 'CELLS for PLATING', combine calculated volumes of cells and media.

• 3.2 Invert the tube 3 × to mix thoroughly.

• 3.3 Label two 96-well plates:

  • Initial Control: C above columns 4–5

  • Treatment Plate: C above columns 1–2, label remaining columns with drug doses.

• 3.4 Transfer cell/media mix into a multichannel reservoir tray.

• 3.5 Use L200 multichannel pipette to dispense 200 µL/well into both plates.

• 3.6 Incubate at 37 °C with 5 % CO₂ for 4–24 h.

PAUSE POINT: Cells can incubate overnight prior to treatment.

• 4. Drug Treatment

• 4.1 Prepare 3 dilutions:

  • 1:100 = 4950 µL media + 50 µL drug

  • 1:20 = 4750 µL media + 250 µL drug

  • 1:5 = 4000 µL media + 1000 µL drug

• 4.2 Filter-sterilize through a 0.2 µm syringe filter into sterile conical tubes.

• 4.3 Label trays with respective dilutions.

• 4.4 Using L20 multichannel pipette, dose treatment plate (exclude columns 1–2).

• 4.5 Return plate to incubator for 72–96 h.

CRITICAL STEP: Confirm control wells reach ∼90 % confluency before proceeding.

• 5. Fixation and Washing.

• 5.1 Aspirate media gently from all wells.

• 5.2 Add 100 µL of 10 % TCA to each well using L200 multichannel pipette.

• 5.3 Incubate at 4 °C for ≥1 hour.

PAUSE POINT: Plates can be wrapped and stored at 4 °C for up to several days.

• 5.4 Wash plates 4 × with tap water. Air dry upside down.

PAUSE POINT: Dried plates may be stored at room temp before staining.

• 6. SRB Staining and Elution.

• 6.1 Add 100 µL of 0.4 % SRB in 1 % acetic acid to each well.

• 6.2 Incubate for 30 min at room temperature.

• 6.3 Rinse vigorously 4 × with 1 % acetic acid.

 Helpful Hint: Dab plate on paper towel to confirm all dye is removed.

• 6.4 Air dry completely upside down.

• PAUSE POINT: Plates can be stored dry before dye elution.

• 6.5 Add 100 µL of 10 mM TRIS base (pH 10.5) to elute dye.

• 6.6 Tap plate gently to mix. Do not invert or shake.

• 7. Plate Reading and Data Analysis

• 7.1 Read absorbance at 570 nm using a microplate reader.

• 7.2 Export data to Excel and enter into SRB Dose Response Template.

• 7.3 Use the following formulas:

  • % Control Growth = 100 × (Aexp – Aini)/(Afin – Aini)

  • SEM = STDEV / √n

  • % SEM = 100 × SEM / (Afin – Aini)

• 7.4 Generate dose-response curve with error bars.

 Helpful Hint: For optimal assay performance, calculate the fold-growth of control cells over96 h. Use the formula ACONfin / ACONini. Fold-growth should be between 5and 10 for valid assays. If below 2, results are unreliable.

Method validation

To demonstrate the effectiveness and reproducibility of the SRB assay, we present representative results obtained from an experiment using nitrosylcobalamin (NO—Cbl) across a range of concentrations (10–100 μM) with A375 human melanoma cells in a 96-well format.

Controls and Assay Validity:

Eight replicates of control wells were used to determine the initial and final optical density (OD) values at 575 nm. The average OD of final control wells was 0.13975 with a standard deviation (SDEV) of 0.0134 and standard error of the mean (SEM) of 0.0033. Treated wells demonstrated fold-growth consistent with assay validity criteria (ACONfin / ACONini > 5).

Percent control growth was calculated using the formula: % Control Growth = 100 × (Aexp – Aini) / (Afin – Aini) Percent SEM was derived as: % SEM = 100 × SEMexp / (Afin – Aini)

Summary of Key Results:

Interpretation:

Percent control growth values below 100 indicate growth inhibition, and values below 0 indicate net cell death. A clear dose-dependent response to NO—Cbl was observed, with maximum cytotoxicity reached at 70 μM and above. The data show high reproducibility across 8 replicates per condition. Assay performance is considered optimal when percent control growth shows a sigmoidal dose-response, with calculated SEMs below 5 %.

Limitations

While the colorimetric cell proliferation assay described here offers a robust and accessible platform for cytotoxicity screening, several limitations should be noted:

• •Adherent Cells Preferred: This protocol is optimized for adherent cell lines. Suspension cell lines can be assessed but will require centrifugation of the 96 well treatment plate prior to fixation.
• •Endpoint Assay: The assay provides a single endpoint measurement of cell mass and cannot distinguish between cytostatic and cytotoxic effects without additional timepoints or complementary assays (e.g., apoptosis markers).
• •Fixed Seeding Density: Although using a standardized seeding density improves ease of use, some fast-growing or slow-growing cell lines may overgrow or fail to reach adequate confluency by the 96-hour mark, affecting assay validity. If that is the case, seeding density to can be changed to 1000 cells per well.
• •Non-Specificity: As a total protein stain, SRB does not discriminate between live and dead cells with residual protein content. This may lead to overestimation of viability in cases of early necrosis or delayed apoptosis. Our assay requires removal of media prior to fixation to overcome this limitation.
• •Environmental Sensitivity: Variability in incubation conditions (e.g., humidity, edge effects, plate reader calibration) can impact data quality. Consistency in plate handling and use of replicates is essential.

Despite these limitations, the assay remains highly useful for medium- to high-throughput applications, especially in resource-limited or training-focused environments.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.