Oct 24, 2024
The VVBlue assay: a plate-readable, dye exclusion-based cell viability assay for the toxicological testing of chemicals
- Marianne Vitipon1,
- Esther Akingbagbohun1,
- Thierry Rabilloud1
- 1Chemistry and Biology of Metals, Univ. Grenoble Alpes, CNRS UMR5249, CEA, IRIG-LCBM, F-38054 Grenoble, France
Protocol Citation: Marianne Vitipon, Esther Akingbagbohun, Thierry Rabilloud 2024. The VVBlue assay: a plate-readable, dye exclusion-based cell viability assay for the toxicological testing of chemicals. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly6z6egx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 22, 2024
Last Modified: October 24, 2024
Protocol Integer ID: 110795
Keywords: Cell toxicology, In vitro toxicology, Cell viability assay, In vitro toxicity testing of chemicals, cell viability assay for the toxicological testing, based cell viability assay, alphazurine viability test, dye exclusion test, toxicological testing, alphazurine assay, viable cell, exclusion by viable cell, cell viability index, dye exclusion, other viability tests such as propidium iodide exclusion, dead cells present in the culture, entering live cell, live cell, dye, number of dead cell, dead cell, other viability test, viability test, assay, toxicity of substance, cell, cell culture well, toxicity, such as pigment, double viability test, cells present in the plate, chemical, crystal violet, testing, cell culture
Funders Acknowledgements:
Agence Nationale pour la Recherche
Grant ID: ANR-21-CE34-0025
Agence Nationale pour la Recherche
Grant ID: ANR-17-EURE-0003
Abstract
A viability test for in vitro cultures, based on the intake of the textile dye Alphazurine A by dead cells and its exclusion by viable cells, is described. This test uses the affinity of Alphazurine A for proteins, so that the dye is retained in dead cells even after rinsing, while its anionic character prevents it from entering live cells. This feature makes this dye exclusion test amenable to a reading in a plate format. The Alphazurine viability test provides an indicator of the absolute number of dead cells present in the culture well. To reach a cell viability index, a “dead cells” control (e.g. cells killed with ethanol) must be added. We also describe a double viability test, which first uses the Alphazurine assay to provide the number of dead cells then a crystal violet assay to provide an index of the number of cells present in the plate. This double test provides a complete appraisal of the situation in the cell culture wells, and has been compared to other viability tests such as propidium iodide exclusion or tetrazolium reduction. Its performances to study the toxicity of substances such as pigments are also established.
Guidelines
Safety concerns
- Acetic acid is an irritant.
- Ethanol is toxic at high doses.
- Crystal Violet is a suspected carcinogen and is toxic for the environment.
- All solutions and wastes used in this protocol should be disposed of respecting applicable local regulations.
Test Setup
The rationale of the alphazurine stain (staining of dead cells in the plate) means that the absorbance reading at 637 nm will provide an index of the number of dead cells. This index must be scaled against wells that will contain only dead cells and thus represent a 100% dead cells level. This means that a plate designed for a complete test shall be arranged as follows
Note
We do not recommend to go below four replicates by condition, which is an absolute minimum.
Readout
The alphazurine stain provides an index of the number of dead cells. Thus, in addition to the desired measurements, the plate(s) must contain wells with all live cells (or the best approximation thereof) and wells with only dead cells. There are therefore three sets of measurements in the plate(s).
A637 all live cells
A637 all dead cells
A637 tested cells
The A all live cells values can be used as a background value, as it integrates the absorbance due to stain adsorbed onto plastic, absorbance of the plate plastic itself and the absorbance due to the few dead cells that may exist in a healthy cell culture.
Then the 100% scale can be calculated with the formula A637 all dead cells - A637 all live cells
Then, for each tested condition, the fraction of dead cells in the well can be calculated by the formula
Fractiondead = (A637 tested cells - A637 all live cells)/ (A637 all dead cells - A 637all live cells)
This number allows to calculate Fractionalive, which is equal to 1- Fraction dead
However, these fractions also depend of the total number of cells that are present at the end of the al-phazurine reading. Depending on how the tested chemicals act on cells, dead cells may still be adher-ent to the plate (which is the case for e.g. ethanol) or dead cells can detach or disintegrate and thus be no longer present in the plate when the alphazurine absorbance is read. To compensate for this phenomenon, the crystal violet staining will provide an index of the total number of cells (either initially dead or alive) that are present at the bottom of the well.
Thus, the fraction of remaining cells is given by the formula
Fraction present = (A590 tested cells/ A590 all live cells)
Thus, the fraction of live cells at the end of the process, compared to the untreated control, is given by the formula:
Fractionalive x Fraction present
Materials
Materials
Culture plates shall be with a transparent flat bottom and treated for mammalian cell adherence. We use 12 or 24 well culture plates from Falcon (#353043Falcon® 12-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid, Individually WrCorningCatalog #353043 and #353047Falcon® 24-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid, Sterile, 50/CasCorningCatalog #353047 , respectively).
Chemicals
Non-denatured ethanol should be used. 96% ethanol is acceptable without correction for titer Acetic acid is 99% pure. We use reference A6283Acetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #A6283 from Sigma Aldrich Alphazurine A was reference A2770 from Sigma Aldrich. This product is discontinued. Alternate references are #ICNA0215473025 from MP biomedicals or reference AC189370100 from ThermoFisher. Crystal Violet is reference C0775Crystal VioletMerck MilliporeSigma (Sigma-Aldrich)Catalog #C0775 from Sigma Aldrich. PBS, calcium and magnesium free, is reference D5652Dulbecco’s Phosphate Buffered SalineMerck MilliporeSigma (Sigma-Aldrich)Catalog #D5652 from Sigma Aldrich.
Equipment
A plate reader able to read absorbance is required. The required wavelengths to be read are 590 nm (Crystal Violet) and 637 nm ( Alphazurine).
Troubleshooting
Plate preparation
Centrifuge cell culture plate for 1200 rpm, 00:02:00 .
Note
Centrifugation prevents any loss of cells.
2m
Remove the medium from well scheduled for 100% dead cells control. Add 300 µL of Ethanol (EtOH) 50% for 00:03:00 -00:05:00 at Room temperature .
Note
The addition of ethanol induces cell death and makes their membranes permeable.
8m
Rinse with DMEM live cell wells.
Note
The medium may have been affected by the 48h culture period or the presence of particles, prompting its replacement with a fresh one.
Remove EtOH 50%. Rinse wells once with clean culture medium. Add a volume of clean culture medium.
Note
The exposure to ethanol dehydrates the cells, necessitating hydration before dyeing them.
Alphazurine dye staining
Add AlphazurineA at a final concentration of 0.2 µL in all wells (50 fold direct dilution from a 10 mg/ml in water).
Note
Alphazurine is added to fresh medium to maintain cells in afavourable environment while staining, ensuring that viability remains uncompromised.
Incubate 00:40:00 at 37 °C .
Note
Incubating cells under cultured conditions ensures that viability remains uncompromised.
40m
Centrifuge 1200 rpm, 00:03:00 .
Note
Centrifugation prevents any loss of cells.
3m
Rinse 3x with 1X Phosphate-buffered saline (PBS).
Note
Rinsing cells with PBS facilitates the removal of dye that have not entered the cells.
Centrifuge for 1200 rpm, 00:03:00 after each rinse.
Note
Centrifugation prevents any loss of cells .
3m
Remove the supernatant.
Note
The last rinse with PBS has to be remove before adding the elution solution.
Elute by adding a volume (equivalent to cell culture medium used) of 50% ethanol 1% acetic acid solution.
Note
Adding acetic acid to ethanol enhances the dye solubility.
Incubate 00:30:00 at Room temperature on a shaker. Centrifuge for 1200 rpm, 00:02:00 .
Note
Centrifugation prevents any loss of cells.
32m
Transfer 200 µL into a 96-well plate.
Note
To eliminate the potential interference from cell absorbance on the original plate, it is advisable to transfer a portion on supernatant into a new plate.
Read absorbance at 637 µL .
Cystal violet dye
1h 23m
Remove the remaining alphazurine eluate. Rinse once with PBS.
Note
The elution solution dehydrates the cells, necessitating hydration before dyeing them with another dye.
Add one volume (equivalent to cell culture medium used) of CV solution at 4 µL in PBS.
Note
As the cells are dead, exposure to water poses no risk to them, so this solution can be prepared using either water or PBS.
Incubate 00:30:00 at Room temperature .
Note
It is not necessary for cells to be under culture conditions for this staining, as all cells are dead.
30m
Centrifuge 1200 rpm, 00:03:00 .
Note
Centrifugation prevents any loss of cells.
3m
Rinse 3x with PBS.
Note
Rinsing cells with PBS facilitates the removal of dye that have not stained the cells.
Centrifuge for 1200 rpm, 00:03:00 after each rinse.
Note
Centrifugation prevents any loss of cells.
3m
Remove the supernatant.
Note
The last rinse with PBS has to be remove before adding the elution solution.
Elute by adding a volume (equivalent to cell culture medium used) of 50% EtOH 1% acetic acid solution.
Note
Adding acetic acid to ethanol enhances the dye solubility.
Incubate 00:45:00 at Room temperature on a shaker. Centrifuge for1200 rpm, 00:02:00 .
Note
Centrifugation prevents any loss of cells.
47m
Transfer 200 µL into a 96-well plate.
Note
To eliminate the potential interference from cell absorbance on the original plate, it is advisable to transfer a portion on supernatant into a new plate.
Read absorbance at 590 µL .