2.12. Sulforhodamine B (SRB) Assay

SRB staining

Materials

- SRB solution (dye): 0.4% SRB (230162, Sigma-Aldrich) in 1% acetic acid diluted with auto ddH₂O

- 0.1% acetic acid (washing buffer): 0.1% acetic acid (695092, Sigma-Aldrich) in auto ddH₂O

- 10% TCA solution (fixation): 10% TCA solution (T0699, Sigma-Aldrich) in auto ddH₂O

- 10mM Tris base solution (elution): Trizma® base (T6066, Sigma-Aldrich) in auto ddH₂O

Procedure

1. Cell seeding in 96well plates (seed a different number of cells depending on their cell sizes)

Example) 1x10³ cells / each well in 96 well plate

2. Drug treatment: serial dilution (total media has to be 200 ul in each well)

Example) 0.01 – 0.1 – 1 – 2 – 5 – 10 – 20 – 40 – 60 – 80 – 100 – 1000 

3. Incubation in 37°C incubator 12 to 16 hours

4. Harvest each plate in once every two days

1) Suction media carefully

2) Put 100 ul of 10% TCA solution in each well by multichannel pipette

3) Incubate the plate in 4°C for 1 h (dark)

4) washing TCA solution by D.W about 3 to 5 times

5) Add 80ul of 0.4% SRB solution using by multichannel pipette

6) Incubate the plate in the dark for 30 min

7) Remove the SRB solution with a 0.1% acetic acid buffer (washing)

8) Dry for 1h

9) Add 150ul of Trizma base solution and shaking 1 h to resolve the staining

10) Measure the absorbance in 540 nm 

11) Analysis the raw data