Crystal Violet Assay - (May/15/2010 )
Hi!
Im trying to use the crystal violet assay for determinate cell viability in drug screening but im having some problems...
I have been used the following protocol:
-Wash plate gently with PBS warmed at least to room temperature
- Carefully remove PBS and add crystal violet solution. Incubate 10 minutes at room temperature
- Wash plate 2x in tap water by immersion in a large beaker. Be careful not to lift off cells. Change tap water between washes.
- Drain upside down on paper towels, than add 1% SDS to solubilize the stain
- Agitate plate on orbital shaker until color is uniform with no areas of dense coloration in bottom of wells
In the step of wash in tap water, i realizing that the cells lift off. Im doing this step in a wrong way?
Someone recomends another protocol? I read some protocols that talk about fix cells in metanol. This will help?
OBS: I work with MCF-7 and HepG2 cell.
OBS 2: Sorry about the bad english 
Your protocol looks fine. The crystal violet should be dissolved in a glutaraldehyde solution so the cells will be fixed.
I found you need to be really really gentle when submerging the plates - I did it in a large sink, and slowly rotated the plates so that the water entered the wells slowly. This was done by inverting the plate to a 45 degree angle, then dipping the bottom edge in the water and slowly lowering the plate until the lower wells were half full, then tipping the plate so that the wells were facing up at a 45 degree angle (to prevent bubble formation), then lowering the plate until the lower wells were full, repeated for the next row of wells.
bob1 on May 16 2010, 09:27 PM said:
I found you need to be really really gentle when submerging the plates - I did it in a large sink, and slowly rotated the plates so that the water entered the wells slowly. This was done by inverting the plate to a 45 degree angle, then dipping the bottom edge in the water and slowly lowering the plate until the lower wells were half full, then tipping the plate so that the wells were facing up at a 45 degree angle (to prevent bubble formation), then lowering the plate until the lower wells were full, repeated for the next row of wells.
Hi bob1! Thanks for your help!
How does glutaraldehyde solution works? Should I use instead the SDS solution?
I used to wash off the excess by adding in a large volume of water with a transfer pipette, swirling and removing. I would do this until the water stayed relatively clear (3-6 times). I'm not sure if this would be more gentle on your cells or not.
Stone on May 16 2010, 04:43 PM said:
bob1 on May 16 2010, 09:27 PM said:
I found you need to be really really gentle when submerging the plates - I did it in a large sink, and slowly rotated the plates so that the water entered the wells slowly. This was done by inverting the plate to a 45 degree angle, then dipping the bottom edge in the water and slowly lowering the plate until the lower wells were half full, then tipping the plate so that the wells were facing up at a 45 degree angle (to prevent bubble formation), then lowering the plate until the lower wells were full, repeated for the next row of wells.
Hi bob1! Thanks for your help!
How does glutaraldehyde solution works? Should I use instead the SDS solution?
SDS would dissolve the cells as it is a detergent. The glutaraldehyde works by cross-linking proteins, much as any other aldehyde fixative will.
bob1 on May 17 2010, 10:31 PM said:
Stone on May 16 2010, 04:43 PM said:
bob1 on May 16 2010, 09:27 PM said:
I found you need to be really really gentle when submerging the plates - I did it in a large sink, and slowly rotated the plates so that the water entered the wells slowly. This was done by inverting the plate to a 45 degree angle, then dipping the bottom edge in the water and slowly lowering the plate until the lower wells were half full, then tipping the plate so that the wells were facing up at a 45 degree angle (to prevent bubble formation), then lowering the plate until the lower wells were full, repeated for the next row of wells.
Hi bob1! Thanks for your help!
How does glutaraldehyde solution works? Should I use instead the SDS solution?
SDS would dissolve the cells as it is a detergent. The glutaraldehyde works by cross-linking proteins, much as any other aldehyde fixative will.
Ah ok! So in what step should I use this solution?
Thanks all for the help!
see attached protocol.
bob1 on May 18 2010, 09:08 PM said:
Bob1
Thanks a lot for the protocol!
