Mitochondria occupy fine astrocyte processes concurrently with astrocyte morphological arborization in vitro. (A) Schematic of astrocyte–neuron co-culture assay. (B) Representative images of rat astrocytes transfected with EGFP (green, top panels) and MitoDsRed (magenta, middle panels) from 4 to 48 h in co-culture with cortical neurons. Inset (bottom panels) of distal astrocyte processes (green) housing mitochondria (magenta) at the leading edge of growing processes (white arrowheads) from 4 to 48 h in culture. Scale bars: 20 μm. (C) Overview of the Seg_Astro image analysis pipeline. (D) Astrocyte secondary, fine/distal, and terminal branch number at 4 vs. 48 h in culture. n = 10–12 cells per time point from one experiment. Unpaired two-tailed t test. (E and F) Total astrocyte mitochondria number and (F) average mitochondrial size in secondary, fine/distal, and terminal astrocyte branches from 4 to 48 h in culture. n = 10–12 cells per time point from one experiment. Data are presented as mean ± SEM. One-way ANOVA with Tukey post hoc test.