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        Crystal Violet Assay

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        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.

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