Cell Enumeration by Crystal Violet Staining

Materials

Staining solution

  • Crystal violet powder: 0.125g
  • Methanol 20%: 50ml ( 10ml methanol + 40 ml H2O)

Lysing solution, 200 ml

  • 0.1 M sodium citrate 5.88 g
  • 50% Ethanol 100 mL : (50ml ethanol (96-99%) + 50 ml H2O)
  • dH2O 100 mL
  • pH 4.2 adjust with HCl

Procedure

  1. Gently aspirate supernatant of cells in a 12-well culture plate
  2. Rinse once with 1ml PBS
  3. Gently add 300ul staining solution to each well.
  4. Incubate at room temperature for 10 min.
  5. Gently rinse 6 times with 1.5 ml PBS.
  6. Treat the stained cell with 350ul lysing solution for 30 min while shaking gently on a rocking shaker.
  7. Dilute cells of the darkest-stained well for the standard curve:
     

    Lysate(ul)

    Buffer(ul)

    100%

    200

    0

    80%

    160

    40

    60%

    120

    80

    40%

    80

    120

    20%

    40

    160

    10%

    20

    180

    0%

    0

    200
  8. Transfer 60 ul of the standard lysates and experimental lysates into 96-well plate, add 240ul of D2O, measure OD at 590 nm. Adjust the diluted fold according to the linear range of standard curve.

Crystal Violet: Sigma, C3886-25G or C3886-100G

Sodium citrate dihydrate : Sigma, W302600-1KG-K

Please see the protocol attached: I made a few corrections. For crystal violet solution you dilute 0.125g of crystal violet in 50 ml of 20% methanol. For lysing solution, dilute 5.88 g of sodium citrate in 100ml of D2O, use HCl to adjust pH to 4.2, add 100 ml of 50% ethanol.

Concerning plates, the protocol is for 12-well plates, which is indicated in the first step. If you want to use 24-well plates just adjust the volumes of staining and lysing solutions accordingly.

We seeded 45 thousand cells per well in 12-well plates for shHK2 experiment. This is in the file describing the experiment we did. Do you have it? It's very important for a student to have a record of the experiments with all the numbers if not in a notebook but at least in the electronic form.

It usually takes cells 6 hours to attach to the bottom of the plate (not the wall). I don't think there is any cell line that can double faster than in 8 hours so it does not make sense to take 6 hours any way - at least 12 hours. If you are comparing different cell lines, I would count time from the time you seeded them. If you are treating cells with something and then looking at the effect of the treatment on cell proliferation then I would count from the time you started the treatment.

Hope this helps.
Masha