Evaluation of cell viability

H/R-induced cell injury was quantified by measurement of lactate dehydrogenase (LDH) activity released from the cytosol of damaged cells in the experimental media⁵³, and by the method of double staining with FDA/PI²¹. At the end of the H/R experiment, 100 μl of cell culture medium were removed and added to a 96 well plate. Then, 100 μl of the reaction mixture (Diaphorase/NAD⁺ mixture premixed with iodotetrazolium chloride/sodium lactate) were added to each well and the plate was incubated for 30 min at room temperature, protected from light. LDH activity was assessed by reading the absorbance of the sample medium at 490 nm in a Victor Multilabe Counter plate reader (Perkin Elmer, Waltham, MA, USA). For FDA/PI staining, cells were plated on glass coverslips and subjected to H/R. Afterwards, cells were treated with 36 μM FDA (Sigma) and 7 µM PI (Calbiochem., San Diego, CA, U.S.A.) for 10 min at 37 °C in PBS. Stained cells were examined immediately with an inverted Zeiss Axiovert 200 microscope (Carl Zeiss, Milan, Italy) and then analyzed. When FDA crosses the cell membrane it is hydrolyzed by intracellular esterases producing a green-yellow fluorescence. Cell damage curtails FDA staining and allows cell permeation by PI that, interacting with nuclear DNA, yields a bright red fluorescence²¹.