MSC coculture inhibits OxPhos inhibition and cytarabine-induced apoptosis with increasing mitochondrial transfer. OCI-AML3 cells were treated with OxPhosi IACS-010759 (20 nM) and/or cytarabine (500 nM) for 72 hours in the presence or absence of MSCs. In cocultured cells, nonadherent OCI-AML3 cells were separated from the MSC monolayer by careful pipetting. After removing nonadherent cells, the OCI-AML3 cells that adhered to cocultured MSCs were trypsinized with MSCs and then separated from MSCs by magnetic-activated cell sorting using anti-CD45 microbeads. (A) Apoptotic cell death of adherent and nonadherent OCI-AML3 cells in single-culture and MSC-coculture conditions was detected by staining with annexin V-FITC/PI and an anti-CD45 APC-conjugated antibody using flow cytometry. Representative flow cytometry plots (bottom) show annexin V (x-axis) and PI (y-axis) staining. (B) The rate of MSC-derived mitochondrial transfer in cocultured nonadherent OCI-AML3 cells was detected by counting the number of GFP and dsRed dual-positive recipient cells per 100 GFP⁺ cells (n > 5) at ×40 magnification by live-cell imaging with confocal microscopy using laser scanning to obtain images. (C) The rate of MSC-derived mitochondrial transfer in OCI-AML3 cells that adhered to MSCs was detected by flow cytometric analysis after depletion of MSCs with MACS as described in supplemental Materials and Methods. Representative flow cytometry plots are shown (right). Error bars in the graphs show the means ± SDs of the results from 3 independent experiments. Two-way analysis of variance (ANOVA) *P < .05; **P < .01.