4.11. Comet Assay

Comet assay was performed under alkaline conditions essentially according to the procedure of Singh et al. [47] with slight modifications. Fully frosted microscopic slides precoated with 1.0% normal melting agarose at about 50 °C (dissolved in Ca²⁺ and Mg²⁺ free PBS) were used. Approximately 10,000 cells were mixed with 75 µL of 2.0% LMPA to form a cell suspension and pipetted over the first layer and covered immediately by a coverslip. The agarose layer was allowed to solidify by placing the slides on a flat tray and keeping it on ice for 10 min. The coverslips were removed and a third layer of 0.5% LMPA (75 µL) was pipetted and coverslips placed over it and kept on ice for 5 min for proper solidification of layer. The coverslips were removed and the slides were immersed in cold lysing solution containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, and 1% Triton X-100 added just prior to use for a minimum of 1 h at 4 °C. After lysis DNA was allowed to unwind for 30 min in alkaline electrophoretic solution consisting of 300 mM NaOH, 1 mM EDTA, pH > 13. Electrophoresis was performed at 4 °C in a field strength of 0.7 V/cm and 300 mA current. The slides were then neutralized with cold 0.4 M Tris, pH 7.5, stained with 75 µL Ethidium Bromide (20 µg/mL) and covered with a coverslip. The slides were placed in a humidified chamber to prevent drying of the gel and analyzed the same day. Slides were scored using an image analysis system (Komet 5.5, Kinetic Imaging, Liverpool, UK) attached to a Olympus (CX41) fluorescent microscope and a COHU 4910 (equipped with a 510–560 nm excitation and 590 nm barrier filters) integrated CC camera. Comets were scored at 100× magnification. Images from 50 cells (25 from each replicate slide) were analyzed. The parameter taken to assess lymphocytes DNA damage was tail length (migration of DNA from the nucleus, µm) and was automatically generated by Komet 5.5 image analysis system.

Treatment of intact lymphocytes with four catechins (C, EC, EGC and EGCG) and the subsequent Comet assay was performed essentially as described earlier by Azmi et al. [48]. For antioxidant study [49], the cells were preincubated with polyphenols in eppendorf tubes in a reaction volume of 1.0 mL. After the preincubation (for 30 min at 37 °C), the reaction mixture was centrifuged at 4000 rpm, the supernatant was discarded and the pelleted lymphocytes were resuspended in 100 µL of PBS (Ca²⁺ and Mg²⁺ free) and layered for further treatment with TBHP (50 µM). The incubation period was 30 min at 37 °C in dark. The other conditions remained the same as described above.