May 21, 2026
Quantitative Cytotoxicity Assessment Using the Sulforhodamine B (SRB) Assay in HCT116 Colon Cancer Cells
- Francisco Stefânio Barreto1,2,
- Andréa Felinto Moura1,2,
- Hândrya Karla Martins Gomes1,
- Manoel Odorico de Moraes Filho1
- 1Laboratory of Experimental Oncology, Drug Research and Development Center, Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazil.;
- 2Graduate Program in Adult Health, Center for Biological and Health Sciences, Federal University of Maranhão, São Luís, MA, Brazil.
- Stefanio Barreto
Protocol Citation: Francisco Stefânio Barreto, Andréa Felinto Moura, Hândrya Karla Martins Gomes, Manoel Odorico de Moraes Filho 2026. Quantitative Cytotoxicity Assessment Using the Sulforhodamine B (SRB) Assay in HCT116 Colon Cancer Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm8dmog3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 21, 2026
Last Modified: May 21, 2026
Protocol Integer ID: 317736
Keywords: sulforhodamine B, cytotoxicity screening, HCT116, cell proliferation, assay in hct116 colon cancer cell, quantitative cytotoxicity assessment, applicable for cytotoxicity screening, stoichiometric binding of srb dye, cytotoxicity screening, colorimetric assay, hct116 colon cancer cell, srb dye, investigating cytotoxic effect, cytotoxicity, cancer cell line, cytotoxic effects in different cell, recovered dye concentration, cell proliferation, neoplastic cell line, total cellular protein content, srb method, mtt assay, removal of the unbound dye, cellular protein, basic amino acid residues of cellular protein, assay
Disclaimer
This protocol was optimized for HCT116 cells and may require adjustments for other cell lines. The authors are not responsible for results obtained under conditions that deviate from those described herein.
Abstract
The sulforhodamine B (SRB) colorimetric assay is a well-established and cost-effective method for evaluating in vitro cell proliferation and cytotoxicity. Since its development, the assay has been widely used as an economical approach for large-scale screening and for investigating cytotoxic effects in different cell-based experimental models. Compared with the MTT assay, the SRB method demonstrates higher sensitivity, improved linearity with cell number, and greater reproducibility across a broad range of neoplastic cell lines. The assay is based on the stoichiometric binding of SRB dye to basic amino acid residues of cellular proteins under mildly acidic conditions. After removal of the unbound dye, the protein-bound fraction is solubilized in a basic solution, and the recovered dye concentration is used to estimate total cellular protein content and, consequently, cell proliferation. The standardized protocol consists of four main steps: preparation of experimental treatments, controlled incubation of target cells, chemical fixation, and SRB staining. The assay produces a stable colorimetric endpoint that is easily visualized and quantified. Due to its low cost, reliability, and simplicity, the SRB assay is widely applicable for cytotoxicity screening in cancer cell lines, pharmacological studies, and molecular biology applications, including the evaluation of gene expression modulation effects.
Guidelines
- Handling information for the HCT116 cell line: follow the ATCC culture guide (https://www.atcc.org/products/ccl-247) or the standardized method in your laboratory.
- All procedures should be performed in a certified biological safety cabinet under aseptic conditions. Users should be familiar with standard cell culture techniques prior to performing this assay. Personal protective equipment (PPE), including gloves, lab coat, and eye protection, must be worn throughout the experiment, especially when handling TCA and DMSO.
Materials
- HCT116 human colon cancer cells
- Culture medium (RPMI or DMEM)
- Fetal bovine serum (FBS)
- Penicillin–Streptomycin solution (1% v/v)
- Sterile phosphate-buffered saline (PBS), pH 7.4
- Trypsin–EDTA solution (0.05%)
- Dimethyl sulfoxide (DMSO)
- Sulforhodamine B (SRB) solution, 0.4% (w/v) prepared in 1% acetic acidTrichloroacetic acid (TCA)
- Acetic acid 1% (v/v)
- Tris base solution 10 mM, pH 10.5
- Milli-Q ultrapure water (sterile)
- 96-well flat-bottom tissue culture plates
- Micropipettes and sterile filtered tips
- Hemocytometer or automated cell counter
- CO₂ incubator
- Biological safety cabinet
- Microplate spectrophotometer (plate reader)
Safety warnings
⚠️ Caution_: Trichloroacetic acid (TCA) is highly corrosive (Sahin et al., 2014). Dimethyl sulfoxide (DMSO) should be handled with care as it can penetrate the skin easily and can decrease the permeability of membranes (Notman et al., 2007). Dispose of all chemical waste according to institutional biosafety regulations.
Ethics statement
The study did not involve human participants or experiments on animals.
Before start
Ensure that HCT116 cells are in exponential growth phase (70–80% confluency) before seeding. Prepare TCA and SRB solutions in advance and store at 4°C. Thaw FBS and allow all reagents to reach room temperature (except TCA, which should remain at 4°C until use). Confirm that the CO₂ incubator is equilibrated to 37°C and 5% CO₂.
HCT116 Cell Culture
Remove the culture medium and wash the cell layer with saline solution.
Add 0.05% trypsin (1x equivalent) and incubate for approximately 2 minutes in a CO₂ incubator. Recommended volumes are 500 µL for T25 flasks and 1 mL for T75 flasks. If cells do not detach completely, gently tap the sides of the flask.
Count the cells using a Neubauer chamber to determine the required volume for the desired number of plates.
For the HCT116 cell line, the recommended density is 4,000 cells/well or 0.4 × 10^5 cells/mL (Zhang et al., 2013).
Dilute the cells in complete medium and adjust the final volume to 11 mL (for one plate).
Dispense 100 µL of the cell suspension into each well.
Incubate the plate overnight in a CO₂ incubator to allow for complete cell adhesion.
Cell Treatment
Time zero control (T0): at the moment of treatment initiation, designate a subset of wells as the T0 control. These wells should be fixed immediately without exposure to any treatment. The T0 control represents the baseline cell biomass at the start of the experiment and is used for growth normalization and data correction.
Prepare stock solutions of the test compounds or experimental treatments at the desired initial concentration using an appropriate solvent (e.g., DMSO, sterile water, or culture medium), depending on compound solubility.
Perform serial dilutions in complete culture medium to obtain the desired range of final concentrations. For dose–response experiments, ensure that the final solvent concentration is kept constant across all treatments, including controls.
Seeded cells (previously plated in 96-well plates and allowed to adhere) should be exposed to 100 µL of each treatment per well, depending on the experimental design.
For serial dilution-based assays, transfer the appropriate volume of the highest concentration into the first well of each row or column and perform stepwise dilutions across the plate, mixing thoroughly at each step to ensure homogeneity and avoid bubble formation.
Controls
Time zero control (T0), as previously described.
Negative control: Cells treated with vehicle only (e.g., DMSO at a final concentration ≤ 0.5% or equivalent solvent used for test compounds).
Blank control: Wells containing culture medium without cells, used for background subtraction.
Positive control (optional but recommended): A known cytotoxic agent (e.g., doxorubicin) at a defined effective concentration.
Incubation
After treatment addition, each well should contain a final volume of 200 µL (100 µL cell suspension + 100 µL treatment solution). Incubate the plates for a specified time (e.g., 72 hours) at 37°C in a humidified atmosphere containing 5% CO₂.
Sulforhodamine B (SRB) Assay
Centrifuge the plate at 1,500 rpm for 5 minutes and remove the medium. Add 100 µL of 10% TCA (4°C) to each well and incubate for at least 1 hour at 4°C to fix cellular proteins. The centrifugation step prior to medium removal is recommended for preserving partially detached or weakly adherent cells after treatment exposure, minimizing cell loss during fixation and washing procedures.
Remove the acid and wash the wells five times with deionized water (Milli-Q), using 200 µL per well.
Remove the deionized water.
Apply 100 µL of 0.4% SRB solution (diluted in 1% acetic acid) to all wells and incubate for 30 minutes.
Remove the SRB and wash five times with 1% acetic acid. Allow the plate to air-dry until no visible moisture remains.
Tris Base Preparation and Absorbance Reading
Dissolve 1.21 g of Tris base in 1 L of Milli-Q water and autoclave the solution.
Solubilize the protein-bound dye by adding 100 µL of 10 mM Tris base buffer to each well.
Homogenize on a shaker for approximately 10 minutes.
Measure the optical density using a spectrophotometer at 564 nm or 570 nm.
Statistical Analysis
Absorbance values will be obtained for the test samples (T), negative control (CN), blanks (B), and time zero (T0), corresponding to cells fixed prior to treatment. Blank values (B) will be used for background subtraction from all experimental conditions prior to analysis.
Cellular responses are classified according to the relationship between T, CN, and T0, following Monks et al. (1991), as stimulation of growth, cytostatic effect, or cytocidal effect.
When T is greater than or equal to CN, the treatment is considered to stimulate cell growth.
When T is greater than or equal to T0 and less than CN, the treatment is considered cytostatic (growth inhibition without net cell loss). In this case, the percentage of cell growth is calculated as: % Cell Growth = 100 × [(T − T0) / (CN − T0)].
When T is lower than T0, the treatment is considered cytocidal (cell death relative to the initial cell population). In this case, the percentage of cytotoxic effect is calculated as: % Cell Death = 100 × [(T − T0) / T0].
All absorbance values will be first corrected by subtracting blank readings (T − B, CN − B, and T0 − B). After normalization, data will be exported and analyzed using GraphPad Prism (GraphPad Software, USA).
Dose–response curves will be generated using nonlinear regression analysis (variable slope model), and IC^^50 values will be calculated. Results will be expressed as mean ± standard error of the mean (SEM) from at least three independent experiments, each performed in technical triplicates. Statistical significance will be determined using appropriate tests (e.g., one-way ANOVA followed by post hoc comparisons), with p c 0.05 considered statistically significant.
Troubleshooting
High variability between replicates: Ensure homogeneous cell seeding and avoid bubble formation during pipetting.
Cell detachment during washes: Reduce washing force and confirm proper cell adhesion before treatment.
High background staining: Increase washing efficiency after SRB staining and ensure complete removal of unbound dye.
Weak staining intensity: Confirm adequate cell density and verify SRB solution preparation.
Edge effect in outer wells: Consider filling peripheral wells with sterile PBS or medium to minimize evaporation.
Protocol references
1. 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;82(13):1107–12. doi: 10.1093/jnci/82.13.1107.
2. Orellana EA, Kasinski AL. Sulforhodamine B (SRB) Assay in Cell Culture to Investigate Cell Proliferation. Bio Protoc. 2016;6(21):e1984. doi: 10.21769/BioProtoc.1984.
3. Bauer JA, Sysel AM, Heston AJ, Dunphy MJ. Standardized sulforhodamine B colorimetric cell proliferation assay for anticancer activity screening in educational and research laboratories. MethodsX. 2025. doi: 10.1016/j.mex.2025.103469.
4. Vajrabhaya LO, Korsuwannawong S. Cytotoxicity evaluation of a Thai herb using tetrazolium (MTT) and sulforhodamine B (SRB) assays. J Anal Sci Technol. 2018;9(1):1–6.
5. Li SZ, Zhang HH, Zhang JN, et al. ALLN hinders HCT116 tumor growth through Bax-dependent apoptosis. Biochem Biophys Res Commun. 2013;437(2):325–330.
6. Monks A, Scudiero D, Skehan P, et al. Feasibility of a high-flux antitumor drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst. 1991;83(11):757–766.
7. Sahin C, Sever C, Aysal BK, Cesur C. An interesting trichloroacetic acid injury in a patient with psychiatric disorder. Int Wound J. 2016;13(2):293. doi: 10.1111/iwj.12254.
8. Notman R, den Otter WK, Noro MG, Briels WJ, Anwar J. The permeability enhancing mechanism of DMSO in ceramide bilayers simulated by molecular dynamics. Biophys J. 2007;93(6):2056–68. doi: 10.1529/biophysj.107.104703.
Acknowledgements
*Francisco Stefânio Barreto and Andrea Felinto Moura contributed equally to the conception, design, and standardization of the protocol. The authors thank the Laboratory of Experimental Oncology (LOE) and the Drug Research and Development Center (NPDM), Federal University of Ceará, for institutional support. This protocol was standardized during the doctoral training of the authors at LOE/NPDM. The authors acknowledge financial support from the National Council for Scientific and Technological Development (CNPq), Brazil, and the Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil.