Preparation of Rat Gingival Mitochondria with an Improved Isolation Method

Figure 1

Diagrammatic representation of the mitochondrial (Mt) preparation procedure from gingival tissue in rats. Tissue specimens were washed and minced with 5.0 mM MOPS buffer (pH 7.4) and then intermittently homogenized at 4°C using a polytron homogenizer for 40, 50, and 60 sec. The homogenized gingival sample was centrifuged at 4,500 g for 10 min, the supernatant was labeled as S1, and the sediment was incubated in a Hanks solution (pH 7.4) containing 0.115%–0.130% (w/v) collagenase at 20°C for 20 min. Following the enzymatic treatment, the homogenized gingival sample was centrifuged at 4,500 g for 10 min. When the supernatant is labeled S2, the sediment was diluted with 50 mM MOPS buffer containing 100 mM KCl, 0.2 mM EDTA, and 0.2% (w/v) BSA and homogenized using a Teflon homogenizer for one min. The homogenate was centrifuged at 600 g for 10 min and the sediment was collected as the nuclear (N) fraction. The supernatant was then centrifuged at 4,500 g for 10 min, the supernatant was labeled as S3, and the sediment was finally collected as the mitochondrial (Mt) fraction.