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, then 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.