cell quantitation: Crystal violet

The crystal violet assay for cell indirect quantification is a simple and quick assay useful for obtaining quantitative information about the relative density of cells in culture.
Crystal violet stains the nucleus dark blue and the cytoplasm light blue as it binds to proteins and DNA.

THE METHOD, IN SUMMARY

In short, the cells are exposed to the dye.

The excess of unbound dye is removed, and finally the bound dye is solubilized and its absorbance evaluated by means of a spectrophotometer, or better, a plate reader.

Since there is a linear relationship, within a certain absorbance range, between the absorbance itself and the concentration of cells – the greater the number of cells, the greater the absorbance- it is possible to have an indirect indication of the number of cells in the growth vessel.

Ad hoc calibration curves, which allow to evaluate the cell density range in which this linear relationship is present, must be foreseen before proceeding with an indirect analysis of the number of cells with these methods.

PROTOCOL ADAPTED FOR 96, 48, 24, 12 AND 6 MULTIWELLS

Plate the cells in a multiwell, leaving at least 3 empty wells (to which you will only add medium) for the blank.
Let the cells grow for the time required (the healthy cells must be well attached to the support)

-eventually treat the cells with toxins or other molecules whose effect on cell growth you are studying, unhealthy cells will detach from the well bottom –

Carefully remove culture medium from wells.
Wash plate gently with PBS warmed at room temperature

multiwell	Volume of PBS to be added
96	100 ul
48	250 ul
24	500 ul
12	1 ml
6	2 ml

Note: When adding the PBS, place the tip of the tip on the edge of the multiwell and drop the PBS, “sliding” it on the edge. Do not pipette the PBS directly onto the cells or you may detach them (e.g., HeLa or 293T cells)

Carefully remove PBS and add crystal violet solution (see at the end of the protocol for the recipe).

multiwell	Volume of Crystal violet solution to be added
96	50 ul
48	100 ul
24	200 ul
12	500 ml
6	1 ml

Note: When adding the Crystal violet solution, place the tip of the tip on the edge of the multiwell and drop the solution, “sliding” it on the edge. Do not pipette the solution directly onto the cells or you may detach them (e.g., HeLa or 293T cells)

Incubate 10 minutes at room temperature on an orbital shaker
Carefully remove crystal violet solution and wash 3 times with PBS (using the same volume specified in the first table)
Invert the multiwell on a napkin to drain all the liquid (the cells are fixed due to the presence of methanol in the crystal violet staining solution)
Add 1% SDS to solubilize the stain

multiwell	Volume of 1% SDS solution to be added
96	200 ul
48	500 ul
24	1 ml
12	2 ml
6	3 ml

note: these latter volumes are suitable for multiwells with epithelial cells at 70-80% confluence. It is necessary to fine-tune the volume to be used and perhaps combine it if the absorbance falls outside the linearity zone of the calibration line

Agitate multiwell on an orbital shaker for 60 minutes, until color is uniform with no areas of dense coloration in bottom of wells.
Read absorbence of each well at 570 nm
Subtract the average absorbance of the wells without cells from that of each well on the plate to subctract backgruound.
The absorbance of the wells without cells, but processed identically to wells with cells, defines the background
of the staining method.

CRYSTAL VIOLET STAINING SOLUTION (0.5%) -100 ML

0.5 g crystal violet powder
80 mL distilled water
20 mL methanol

Dissolve crystal violet powder in water and then add methanol.

Store solution in the dark at room temperature.

Use within 2 mo.

1% SDS SOLUTION – 500 ML

5 g SDS

500 ml water

Dissolve SDS in water

Store at room temperature

CALIBRATION CURVE

To construct a calibration curve to evaluate the values within which there is a linearity between the number of cells in the well and the absorbance of the crystal violet solubilized, it is necessary to prepare a multiwell containing serial dilutions of cells plated at a certain concentration, which must be adhered and grow and absorbance must be evaluated the next day.

For example, considering a 24 multiwell, you can plate 100000 or 160000 cells in the first column and then make a serial dilution (1:2) in the adjacent well