Crystal violet and State's cell - (Jan/24/2007 )

Hello guys,

I realise some infection in macrophage and I want to see the effect of infection on cell viability. I read in a paper that it's possible to fix the cell with 10% formalin (10 to 15 min) and after stained the cell with crystal violet 0,13% (>2h). After this time you can read the absorbance.
Is someone know this protocol? Can you explain the exact protocol and how does it works?

Thanks a lot

biofred

Crystal violet (or Gentian violet) has the ability of binding DNA, so you can easily visualize cells nuclei after staining.

1. Wash with PBS and fix cells with a suitable fixer (10% formalin or 15% formaldehyde at least 10 min or overnight at 4ºC works good).
2. Wash 3 times with PBS.
3. Add 0.1% CV (dissolve in water and filter before use). Incubate at RT at least 15 min.
4. Discard CV excess and wash with deionizated water until no violet color realease from culture plate (soft washes).
5. Add 33% acetic acid to solubilize CV (optional)
6. Read absorbance at 595 nm or count cells under microscope

Good luck!

Hello

Thanks a lot for the protocol.
But You said that crystl violet can bind DNA but I read that crystal violet can differenciate adherent cell vs non adherent cell. What the link between adherence and binding DNA??? Do you know?

biofred

Hi, biofred.

Actually I don't know if CV can differentiate adherent cells, but I've seen other protocols where CV is applied to cells after several washes too, so some attached cells will detach from surface and then CV will stain only attached cells.

Hi Biofred,
Iam also using same method. you also can use its.Protocol is following
First after infection and incubation wash cells with PBS and then fix cells by 100% methanol for one min.
Then add .75% crystal violet strain for each well for 5 min.
remove its wash cells with PBS for removing nonspecific binding
and finally dissolved in 1% SDS and take reading at 595 nm
and calculate % growth, cytotoxicity by following equation
% growth= (sample reading/controll reading)*100
Cytotoxicity= 100-% growth.
all the best and good luck too
awadh