Wittrup: Crystal Violet Toxicity Assay Protocol
Subculture Cells
- Determine optimal number of cells for staining after 4 days
- The cell density must be very low for proper analysis
- For LS174T cells, ~2500 cells/well in flat bottom 96-well plate is ideal
- Day 1: Subculture and allow cells to attach to plate overnight
Incubate Cells
- Day 2: incubate cells with appropriate concentration of toxin (e.g. dilution series of toxin, combining toxins, etc.)
- Include blank wells for background and control wells with no toxin for normalization
- Incubate at 37 C in 5% CO2 for 3 days
Toxicity Assay Plate Staining
- Day 5: remove media w/ multi-well pipettor
- Add 50-100 μL of Cytofix and incubate 30 min on ice
- Remove Cytofix and add 100 μL of Crystal Violet Stain (include blank wells for background)
- Incubate 10 min at RT
- Remove Crystal Violet Stain
- Wash 2X w/ multi-well pipettor (100 μL of water)
- Wash 2X w/ water (rinses sides of wells to remove stain)
- Extract dye from live cells with 100 μL Sorenson’s buffer
- Incubate 30 min at RT on rotating shaker
- Take A540 on plate reader
Data Analysis
- Subtract the background wells from the absorbance readings
- Divide the experimental wells by the control wells (no toxin) to normalize the data
Crystal Violet Stain (200 mL)
0.5% Crystal Violet (toxic) 1 g
20% Methanol 40 mL
dH2O 160 mL
Sorenson’s Buffer (200 mL)
0.1 M sodium citrate 5.88 g
50% Ethanol 100 mL
dH2O 100 mL
pH 4.2
