Extended Data Fig. 4: Identification and quantitative analyses of in vivo-generated mitophers by FACS.
(a–k) FACS analyses, TEM images and microscopic analyses showing the identification of mitophers according to the combination of three features: diameter, presence of the interior mitochondrion, and externalized phosphatidylserine (PS) on the mitopher membrane. (a) Shows the procedures for dissecting worms. (b–d,h) Show the gating strategies. The forward scatter signals (FSC) from the reference microspheres (Beads, 0.45 μm, 0.5 μm, 1 μm, 2 μm, 3 μm) were used to estimate the size range of samples (b) and according to the size, the sample was gated into four groups (c). Annexin V with Alexa Fluor 647 was used to label the externalized PS, and MTG was used to label mitochondria. A representative TEM image (e) and the corresponding bar graph (f) indicated that structures from R2-Q2 region in (d) were all mitophers (n = 21). Confocal images indicate that the mitophers displayed Annexin V signals on the surface and interior MTG signals (g). The structures from the R1-Q3 region were all spermatids, confirmed by TEM analysis (i,j) and the DIC microscopy analyses (k). (l,m) Bar graph and representative image showing that approximately 290 sperm per male (n = 8 biologically independent experiments) and 295 sperm per male (n = 14 biologically independent worms) were detected by FACS and microscopic imaging, respectively. Each dot represents the spermatids number in one male worm. (n) Bar graph showing the total number of mitophers per male in a day-1 adult N2 male. Each dot represents the average mitopher number per male in one experiment. (o) Bar graph indicating the number of extracellular mitophers normalized to the number of spermatids in N2 males. n = 40,000 spermatids were analysed. Each dot represents the average number of mitophers normalized to one spermatid in one independent experiment. The number of biologically independent experiments is 3 in (a–k), 6 in (n), 8 in (o). Source numerical data (f,j,l,n,o) are available in source data.
