316 Gut and Liver, Vol. 5, No. 3, September 2011 Two biopsies were also taken for each comet assay. Biopsies for the comet assay were placed immediately into Dulbecco’s modified Eagle Medium DMEM (Gilbico Ltd., Paisley, Scotland) and stored at 48°C until processed. The delay between biopsy and assay varied but was never longer than 24 hours. The study was approved by the local Research Ethics at hospital. 1. Preparation of the single-cell suspension The comet assay procedure is as follows: Two biopsies were suspended in 800 mL of DMEM with 100 mL of 0.1% pronase (Sigma Chemical Co., St. Louis, MO, USA) and 100 mL of 0.03% collagenase (Sigma Chemical Co.), and incubated at 37°C for 25 minutes with gentle shaking. Incubation was limited to 100 minutes to avoid complete digestion of the biopsies, the aim be- ing to release cells predominantly from the epithelial layer. At the end of incubation, the biopsy remnants were removed, the cell suspension centrifuged at 2,400×g for 7 seconds, and the supernatant was discarded. The yield of cells released from the biopsy following enzyme digestion was determined by staining 50 uL aliquots of the cell suspensions with 150 uL methyl green followed by microscopic examination in a counting chamber. 2. The comet assay The comet assay was performed according to Singh’s original description with minor modifications.5 Slides were coated with a thin film of 1% w/v normal melting point agarose dissolved in phosphate buffered saline (PBS). Low melting point agarose (300 mL of 1% w/v in PBS, kept at 37°C) was added to 60 uL cell suspension at room temperature, giving a final agarose concentration of 0.83% w/v and an average concentration of cells in the agarose of 0.37×105/mL. 162 uL of cells suspended in agarose was aliquoted onto each of the two slides and cov- ered with a 22×32 mm cover slip (162 mL had been determined previously as the volume that gave the appropriate gel thickness and cell density beneath a cover slip of this size). Slides were placed on a cold block to allow the agarose to set, and then the cover slips were removed. All subsequent steps were carried out in subdued lighting to prevent DNA damage from UV light. Slides were placed in lysis buffer (2.5 M NaCl, 100 mM Na EDTA, 10 mM Tris pH 10, and 1% Triton 2 [Sigma Chemical Co.] at 48°C for 1 hour. They were then placed in running buffer (300 mM NaOHq1mM EDTA, pH 13, 48°C) for 20 minutes to allow DNA unwinding, followed by 15 minutes electrophoresis at a constant voltage of 23 V (0.36 V/cm, 310-315 mA). These electrophoresis conditions had been identified in prior experiments as giving a suitable range of DNA damage in gastric cells. Afterwards, slides were carefully removed from the tank and placed in a dish with ice cold neu- tralising buffer (400 mM Tris pH 7.5, for 5 minutes). Slides were dried gently and stained with 25 mL of 20 mlrml Ethidium Bro- mide (Sigma Chemical Co.). By using a fluorescence microscope, count of 100 cells was carried out at least twice for each slide and the percentage of cells that were comets was recorded (Figs 1 and 2). 3. Statistics For comparisons of parametric data, t-tests are used and 95% confidence intervals are expressed. Two-tailed p-values were calculated in all cases; a value of less than 0.05 is taken to im- ply statistical significance. Calculations were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). RESULTS 1. Comet percentage (DNA damage) in gastric cells of 19 normal individuals and 19 patients of gastritis under dif- ferent incubation times In Tables 1 and 2, we compared the percentage of DNA dam- aged gastric epithelial cells in seven normal individuals with Fig. 1. Fluorescent photomicrograph showing a comet. The fluores- cent core is located in the centre of the picture, with a zone of fluo- rescence extending towards the anode. Fig. 2. Fluorescent photomicrograph showing two normal nuclei with no DNA damage.