Mitochondrial transfer enhances recovery after neural damage. (A) Schematic illustration of experimental timeline. (B) MitoTracker-labeled astrocytic mitochondria transferred into neurons (TUJ1-positive cells). (C-E) After differentiation day 7 neurons were induced with 100 μM H2O2 for 3 h to show neurotoxic damage, cells were treated with isolated astrocytic mitochondria twice. Immunofluorescence staining (C) and quantification of TUJ1-positive neurons (D) are shown. (E) RT-PCR analysis of Tuj1 mRNA in mitochondria-treated cells. Gapdh expression was used as a loading control. (F) Comparison of different methods of mitochondrial isolation. Mechanical crushing was performed 30 times/30 s off/30 times/30 s off/30 times and sonication was performed 5 s/5 s off/5 s. Error bars, standard error (S.E.). **P < 0.01. Scale bar, 20 μm. TUJ1, class III beta-tubulin; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcription-polymerase chain reaction.