Cycle test kit

Flow cytometric detection of sperm DNA chromatin damage was made according to the method as described by Martinez-Soto et al. [12 (link)]. It depends on the fluorescence emission from sperm cells stained with propidium iodide (PI) that binds to DNA. The semen sample were diluted with phosphate buffered saline (PBS) to 2 × 10⁶ sperm/mL. Fifty μL of semen sample was directly stained with 50 μg/mL PI, using the cycle test kit (Becton Dickinson, USA); PI was mixed with the semen and analyzed immediately by FACSCalibur flow cytometry with CellQuest software (Becton Dickinson Biosciences, USA). Ten thousand events were measured for each specimen; this permitted state of condensation of the sperm chromatin was analyzed, as the DNA condensation is directly related to PI uptake. The geometric mean fluorescence intensity (GMFI) was used to measure the degree of sperm DNA staining with PI. The sperm with altered nuclear condensation (DNA decondensation and fragmentation) takes more stains (Figure 2). These tests were performed on patients before and 3 months after varicocelectomy.

Cell viability was measured using the WST-8 assay (Dojindo, Kumamoto, Japan). To detect apoptosis, flow cytometry (FACSCalibur; BD) was used after propidium iodide staining with a Cycle Test Kit (BD), and the number of cells in the sub-G1 stage was calculated using FlowJO (Tomy Digital Biology, Tokyo, Japan). In addition, TUNEL assay was performed to detect apoptosis in the cells transfected with BMCC1 or GFP expression vectors, or with BMCC1 siRNAs. Cells were seeded onto coverslips and stained using the In situ Cell Death Detection Kit, TMR red (Roche). To isolate mitochondria, cells were lysed in a fractionation buffer (20 mM HEPES-HCl (pH 7.5), 10 mM KCl, 15 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose), homogenized and centrifuged for 10 min at 800 × g. The supernatant was centrifuged for 15 min at 10 000 × g and divided into cytoplasmic (supernatant) or mitochondrial (pellet) fractions.