|
1. Prepare the cell (6, 12, or 24 well plate) for the assay.
2. Add to ¼ volume of cold 50% TCA on top of the medium (final 10 %), to fix the cells.
3. Incubate plate for no more than 60 minutes at 4 °C. Aspirate TCA and rinse the wells with water 5 times to remove TCA and others.
4. Plates are air dried and stored until use. (1st Stopping Point)
5. Add sulforhodamine B Solution (0.4% in 1% acetic acid) in an amount sufficient to cover the well surface (e.g., 250 microL per well (24 well plate).
6. Allow cells to stain for 20-30 minutes.
7. Wash cells with 1% acetic acid, until unincorporated dye is removed (5 times).
8. Air-dried at room temperatures for 30 min. ( 2nd stopping point ; samples are good up to 3 months).
9. Solubulize bound sulforhodamine B dye with 10 mM Tris pH=10.5 for 5 minutes at room temperature (e.g., 2ml per well in 24 well plate).
10. Shake the dish on an orbital shaker (the solubulized red color to be read).
11. Measure absorbance at a wavelength of (490-530 nm). Dilute the samples with the tris buffer, if red color is too intense.
|
