PMC

ROS measurement. Young, middle, and old represent (A) 20,18, and 19 muscle or (B) 19, 20, and 20 synaptosomal cell lines in different cybrid groups, respectively. Mitochondrial superoxide level was measured with MitoSOX red mitochondrial superoxide indicator (A, B). Three determinations were made.

Growth test. Young, middle, and old represent 24,20, and 25 muscle (left) or 29,31, and 69 synaptosomal (right) cell lines in different cybrid groups. Cells were grown in glucose DMEM for 24 h and then (A, D) in galactose medium or (B, E) in glucose DMEM for another 72 h. (C, F) The growth index was calculated as indicated.

The complex-dependent respiration measurement. Young, middle, and old represent 10,10, and 10 muscle (left) or 10,10, and 15 synaptosomal (right) cell lines in different cybrid groups, respectively. About 5×10⁶ cells were treated with digitonin before measurement. The substrates for measuring (A, D) complex I-dependent, (B, E) complex Ill-dependent, and (C, F) complex IV-dependent respirations are described under Materials and methods.

ATP content analysis. Young, middle, and old represent (A, B) 20, 18, and 19 muscle or (D, E) 19, 20, and 20 synaptosomal cell lines in different cybrid groups, respectively. The (A, D) total and (B, E) oligomycin-resistant ATP content was measured. The ATP/ADP ratio was also measured in a separate experiment. Young, middle, and old represent (C) 10,10, and 10 muscle or (F) 9, 9, and 9 synaptosomal cell lines in different cybrid groups, respectively. Three determinations were made.

Cell viability determination. Young, middle, and old represent 24, 20, and 25 muscle (left) or 29, 31, and 69 synaptosomal (right) cell lines in different cybrid groups. Cells were grown in glucose DMEM for 24 h and then (A, D) in galactose medium or (B, E) in glucose DMEM for another 72 h. (C, F) The viability index was calculated as indicated.

Endogenous respiration measurement. Young, middle, and old represent 24, 20, and 22 muscle (left) or 29, 31, and 68 synaptosomal (right) cell lines in different cybrid groups. Respiration was measured (A, F) in intact cells and in cells treated with (B, G) oligomycin or with (C, H) FCCP. The activities were determined using about 5 × 10⁶ cells. The ratios of various respiration rates were calculated as indicated in (D, E, I, J). (K) A representative trace of oxygen measurement to demonstrate how we obtained the base, uncoupled, and maximal respiration rates.