I am doing proliferation assay, after i fix the cells with 4% paraform aldehyde, i stain the cells with Crystal Violet for 2 hrs, after washing excessive dye i air dry my plates and then use 10% acetic acid and measure the OD at 600nm.
I am confused will this 10% acetic acid just remove the stain from the cells or will it detach the cells completely from the plate as i am working with adhesive mammalian cells.
Akshamal is right, essentially the acetic acid solubilizes the stain. During crystal violet staining in short you are washing off non-adherent (dead) cells before applying the dye. Crystal violet is then applied and binds to DNA and protein of the remaining (attached) cells. After drying when solubilizing the dye, I add methanol and incubate on a rocker (low speed). The cells at this point are dead. So I am not sure what you mean by the latter part of you're question. The only purpose of the solvent is to resolubilize crystal violet for spectrophotometry. If cells are detached, this should not significantly affect OD readings at 570nm. If you are concerned about detached cells affecting OD readings, run negative control wells with cells that are seeded, treated, washed etc. but are not stained with dye but are subject to all other steps of the protocol (washes, 10% acetic acid). Use these then instead for background subtraction rather than unseeded wells. This assumes that the background OD from your protocol is just that of the plastic and solvent (and not reflective of residual dye left after washes). Attached is a cold spring harbor protocol that I've used in the past working with rat glioma cells.
Akshamal is right, essentially the acetic acid solubilizes the stain. During crystal violet staining in short you are washing off non-adherent (dead) cells before applying the dye. Crystal violet is then applied and binds to DNA and protein of the remaining (attached) cells. After drying when solubilizing the dye, I add methanol and incubate on a rocker (low speed). The cells at this point are dead. So I am not sure what you mean by the latter part of you're question. The only purpose of the solvent is to resolubilize crystal violet for spectrophotometry. If cells are detached, this should not significantly affect OD readings at 570nm. If you are concerned about detached cells affecting OD readings, run negative control wells with cells that are seeded, treated, washed etc. but are not stained with dye but are subject to all other steps of the protocol (washes, 10% acetic acid). Use these then instead for background subtraction rather than unseeded wells. This assumes that the background OD from your protocol is just that of the plastic and solvent (and not reflective of residual dye left after washes). Attached is a cold spring harbor protocol that I've used in the past working with rat glioma cells.
Thank you so much for your responses. They are quite helpful.
Regards
Dear Ali, stained crystal violet will be extracted or eluted with 10% acetic acid.
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