Sulforhodamine B (SRB) Cell Cytotoxicity Assay

Introduction

Sulforhodamine B (SRB) is a bright-pink aminoxanthene dye with two sulfonic groups that can form an electrostatic complex with basic amino acid residues in slightly acidic conditions, but it can dissociate under basic conditions. Since the binding of SRB is stoichiometric, the incorporated dye released from stained cells after washing is directly proportional to the cell mass and can be measured at 565 nm.

Sulforhodamine B (SRB) cell cytotoxicity assay has been widely used for drug toxicity screening and cell proliferation detection against different types of cancerous and non-cancerous cell lines. In addition, this assay does not depend on the metabolic activity of the cells and therefore shows less interference with the testing compounds. The protocol described here has been optimized for the toxicity screening of compounds to adherent cells in 96-well format.

Features

• Sensitive
• Easy-to-use
• Reproducible
• Great linearity
• Stable end-point
• Good signal-to-noise ratio

Application

• High-throughput drug screening
• In vitro cell toxicity and proliferation studies

Materials

• Appropriate culture medium
• Trypsin solution
• Dimethyl sulfoxide (DMSO)
• Fixation solution
• Washing solution
• Solubilization solution
• SRB dye solution
• Doxorubicin
• Phosphate buffer solution (PBS)
• 96-well plate
• Multi-well spectrophotometer

SRB Cell Cytotoxicity Assay Procedure

• Cell culture
  1) Remove medium and wash the cells once with sterilized PBS.
  2) Trypsinize and spin down the cells.
  3) Add 5 mL of growth medium to disperse the cells and determine the cell density by using a hemocytometer.
  4) Add growth medium to adjust the cell concentration to obtain an appropriate seeding density.
  5) Add 200 µL of the cells with a recommended density between 5,000-20,000 cells/well to a 96-well plate.
• Compound treatment
  1) Prepare serial dilutions of your testing compounds appropriately by using DMSO as solvent and add compounds to the experimental wells.
  2) Prepare a DMSO-only well as control, and another well containing culture medium-only as background control. In addition, add 1 µL of 20 mM doxorubicin to a well containing the cells as an inhibitor control.
  3) Incubate the plate at 37°C in a humidified incubator with 5% CO₂ for 72 hours.
• Cell fixation
  1) Without removing the culture supernatant, add 50 µL of the fixation solution to each well.
  2) Incubate the plate for 1 hour at 4°C, then remove the solution and wash the wells 3 times.
  Note: Washing should be done as gentle as possible to avoid disturbance of the cell monolayer.
  3) Remove wash solutions as much as possible by pipetting.
  Note: After cell fixation, washing and drying steps are complete, the plate can be stored at room temperature for a month if desired.
• SRB staining
  1) Add 45 µL of SRB solution to each well and stain for 15 min at room temperature in the dark.
  Note: SRB should be protected from light as it is light sensitive.
  2) After incubation, remove the staining solution and add 200 µL of washing solution to wash for 4 times.
  Note: Washing should be done as quickly as possible to avoid bleaching.
  3) Remove wash solutions as much as possible by pipetting and air-dry the plate if necessary.
• Solubilization
  1) Add 200 µL of solubilization solution to each well.
  2) Shake the plate or place the plate on a shaker for 10 min at room temperature.
• Measurement
  1) Measure the OD at 565 nm.
  2) If intense color is observed (> OD 3.5), you may use a suboptimal wavelength (e g. 490-530 nm) to lower the readings back to the linear range of your instrument.
• Calculation
  1) Correct calculation by subtracting the OD of the control containing only the culture medium (background control well) from all samples readings.
  2) Calculate the percentage of cytotoxicity using the formula below:

References

1. Orellana E. A. et al.; Sulforhodamine B (SRB) assay in cell culture to investigate cell proliferation. Bio Protoc, 2016, 6(21).
2. Vichai V. et al.; Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc, 2006, 1(3): 1112-1116.

For research use only. Not for any other purpose.