Crystal Violet Assay

This is a simple assay useful for obtaining quantitative information about the relative density of cells adhering to multi-well cluster dishes. The dye in this assay, crystal violet, stains DNA. Upon solubilization, the amount of dye taken up by the monolayer can be quantitated in a spectrophotometer or plate reader.

1. Carefully remove culture medium from wells.
2. Wash plate gently with PBS warmed at least to room temperature:

   Number of wells	Volume
   96	0.2 mL
   48	0.5 mL
   24	1 mL
   12	2 mL
   6	3 mL

3. Carefully remove PBS and add crystal violet solution . Incubate 10 minutes at room temperature:

   Number of wells	Volume
   96	50 uL
   48	100 uL
   24	200 uL
   12	500 uL
   6	750 uL

4. Wash plate 2x in tap water by immersion in a large beaker. Be careful not to lift off cells. Change tap water between washes.
5. Drain upside down on paper towels, than add 1% SDS to solubilize the stain:

   Number of wells	Volume
   96	100 uL
   48	300 uL
   24	600 uL
   12	1 mL
   6	1.5 mL

6. Agitate plate on orbital shaker until color is uniform with no areas of dense coloration in bottom of wells.
7. Read absorbence of each well at 570 nm.