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Cell viability determination using crystal violet staining

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1. Background and purpose of the procedure

Adherent cells detach from cell culture plasticware during cell death. This feature is used to indirectly quantify cell death or determine changes in proliferation after stimulation with pharmacological or radiological agents. To quantify adherent cells, we stain attached cells with crystal violet, a dye that bind to proteins and DNA. Dead cells lose their adherence to the plasticware and are subsequently lost from the total cell population thus, reducing the total crystal violet staining.

2. Materials and equipment

  • Adherent cells treated with pharmacological or radiological agent
  • D-PBS, without Ca++ and Mg++ (Gibco, 14190144)
  • Crystal violet staining solution (see 3. Recipes)
  • Lysing solution (see 3. Recipes)
  • Flatbed scanner
  • 96-well plate reader for absorbance measurement at 570 nm
  • 96-well plate, flat bottom
  • Bench rocker
  • Absorbent paper
  • Vacuum pump

3. Recipes

Crystal violet staining solution

0.5% Crystal violet dye (Fisher Chemical, Certified Biological Stain, C581-100) [0.5 g for 100 mL]

20% methanol (Fisher Chemical, Certified ACS, A412P-4) [20 mL for 100 mL]

ddH2O qsp 100 mL [80 mL for 100 mL]

Dissolve crystal violet first in water and then add methanol. Solution can be stored in the dark at room temperature for up to 3 months.

Lysing solution

0.1 M Sodium citrate dihydrate (Fisher Chemical, Certified, S279-500) [5.88 g for 200 mL]

25% Ethanol 200 proof (Decon Labs, 2701TP) [50 mL for 200 mL]

ddH2O qsp 200 mL [150 mL for 200 mL]

Adjust pH to 4.2 using 12 N HCl (Fisher Chemical, Certified ACS Plus, A144-500)

4. Methods

  1. Gently aspirate the cell culture media from the tissue culture plate or dish
  2. Rinse cells using D-PBS without Ca++ and Mg++
  3. Gently add crystal violet staining solution [300 µL for 12-well plate, 500 µL for 6-well plate]
  4. Incubate at room temperature for 10 to 15 min on a bench rocker
  5. Aspirate the crystal violet staining solution with a disposable plastic pipet, be careful not to disturb cells
  6. Rinse the plate/dish by immersion in a large tray containing tap water
  7. Discard water
  8. Repeat rinses until the water remain colorless after plate immersion
  9. Invert the plate/dish on absorbent paper and tap gently to remove any liquid
  10. Air-dry the plate without its lid for 2 hrs at room temperature or 35-45 min in a dry chamber at 37˚C
  11. Place the plate/dish on the flatbed scanner and scan the plate/dish
  12. Add Lysing solution [350 µL for 12-well plate, 600 µL for 6-well plate]
  13. Incubate at room temperature for 30 min on a bench rocker
  14. Transfer 60 µL of the lysate into a flat bottom 96-well plate [dilute with 140µL of ddH2O if needed]
  15. Measure the optical density at 570 nm with an absorbance plate reader

5. References

Feoktistova M, Geserick P, Leverkus M. Crystal Violet Assay for Determining Viability of Cultured Cells. Cold Spring Harb Protoc. 2016 Apr 1;2016(4):pdb.prot087379. doi: 10.1101/pdb.prot087379. PMID: 27037069.

Cell Enumeration by Crystal Violet Staining protocol from Xin Chen Lab at UCSF School of Pharmacy

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