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