| in vitro |
Sulforhodamine B colorimetric assay in cell culture to analyze cell proliferation
1. Prepare treatment solutions with sufficient volume for triplicates in 96-well plates (50 μl per replicate) or six replicates in 384-well plates (10 μl per replicate). Ensure volumes account for pipetting variations.
2. Treatment solutions can be prepared in either aqueous solution (e.g., Opti-MEM for transfections) or a suitable solvent (e.g., DMSO).
3. Remove the medium from cell monolayers and wash cells once with sterilized PBS. Add 1 ml (for 100 mm plates) of 0.25% trypsin to cover the cell growth surface evenly.
4. Incubate at 37 °C for 5 minutes or until cells start to dissociate. Inactivate trypsin with 10 volumes of culture medium containing FBS. Mix up and down to obtain a homogeneous single-cell suspension.
5. Transfer the cell suspension to a sterile Falcon tube.
6. Determine cell concentration using a hematocytometer chamber with a 1:1 mixture of cell suspension and 0.4% trypan blue solution. Optionally, spin down cells before counting to wash trypsin and resuspend in a growth medium.
7. Adjust cell concentration with growth medium (10% FBS) for appropriate cell seeding density per well.
8. Mix treatment solutions and dispense 50 μl (96-well format) or 10 μl (384-well format) into each well.
9. Thoroughly mix the cell suspension and add 50 μl (96-well format) or 10 μl (384-well format) to each well with treatment solutions.
Note: Ensure even cell distribution in the well bottom, avoiding shaking to prevent 'ring effects.
10. Set aside three wells for untreated or vehicle control and three wells for background subtraction and incubate the plate at 37 °C with 5% CO2 until ready for reading.
11. Add 25 μl (96-well format) or 5 μl (384-well format) of cold 50% TCA directly to medium supernatant. Incubate plates at 4 °C for 1 hour without mixing.
12. Wash plates four times by submerging in slow-running tap water, and tapping excess water into a paper towel. Allow the plate to air-dry at room temperature.
13. Add 50 μl (96-well format) or 20 μl (384-well format) of 0.04% SRB solution to each well.
14. incubate at room temperature for 1 hour and Rinse plates four times with 1% acetic acid (200 μl for 96-well format or 30 μl for 384-well format). Allow the plate to air-dry at room temperature.
15. Add 50 μl to 100 μl of 10 mM tris base solution (pH 10.5) to each well and shake the plate on an orbital shaker for 10 minutes to solubilize the protein-bound dye.
16. Measure the absorbance at 510 nm in a microplate reader.
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