Wittrup: Crystal Violet Toxicity Assay Protocol

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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