A Silver Staining Method for Single-cell Gel Assay

Abstract

S U M M A R YThe single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. T HE single-cell gel assay (also termed comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. The technique was developed to visualize the DNA damage induced by radiation in mammalian cells (Ostling and Johanson 1984). This method has many applications in radiation biology, in estimation of oxidative damages and DNA crosslinks, in apoptosis, and in genotoxicity induced by chemical compounds (McKelvey-Martin et al. 1993). A small number of cells are immersed in an agarose gel, lysed, subjected to an electrophoretic field, and then stained with a DNA-binding fluorescent dye. The broken DNA fragments, negatively charged, migrate towards the anode and the cells can be observed under a fluorescent microscope for estimation of the damaged DNA that forms a tail like a "comet." The quantity of DNA separated from the head of the comet is proportional to the dose of irradiation.Several versions of the comet assay are in use in research laboratories, and there are also commercial kits. There are two forms of comet assays: the neutral method for the detection of DNA double-strand breaks, and the alkaline method, which detects DNA single-strand breaks and alkali-labile lesions (Fairbairn et al. 1995). Usually, the comets are visualized and evaluated with fluorescent DNA stains, such as propidium iodide or ethidium bromide (potential carcinogens). These DNA stainings require a high-quality microscope with epifluorescent optics, a 100-W mercury lamp, a sensitive CCD camera light, and sophisticated image analysis software. Unfortunately, the slides can not be stored for long periods of time (the dye is bleached out within a day) so they must be analyzed immediately (an alternative is to store the slides with the gels and re-stain them when reexamination of the samples is needed).We are working with lymphocytes from the peripheral blood of cancer patients to evaluate the effect of cytotoxic drugs. Cells are analyzed before and after chemotherapy. We have developed a silver staining of the comets that allows long-term storage and retrospective comparative evaluation of the cells before and after therapy. We tested a published silver staining method (Trevigen, Inc. 1999) but obtained a very low sensitivity (data not shown). Here we show the modifications introduced to the silver staining metho...

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Correlation of the cytotoxic activity of four different alkaloids, from Chelidonium majus (greater celandine), with their DNA intercalating properties and ability to induce breaks in the DNA of NK/Ly murine lymphoma cells

“…After that, the slides were washed with distilled water, dried for 30 min at 37 • C, fixed for 10 min in a solution of 15 % (w/v) tri-chloroacetic acid, 5 % ZnSO 4 and 5 % glycerol, and then dried. The DNA comet pattern was employed, using the modified silver staining procedure described in [13]. Staining solutions were freshly prepared before each staining procedure by mixing 7 ml of solution A (0.3 % ammonium nitrate, 0.3 % silver nitrate, 0.7 % tungstosilicic acid, 0.6 % formaldehyde) with 14 ml of solution B (5 % sodium bicarbonate).…”

Assessment of DNA Damage in Leukocytes of Patients With Coronary Artery Disease by Comet Assay

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