Xfp 8 well miniplates

Mouse striatal HdhQ7/Q111 cells were seeded in XFp 8-well miniplates (103025-100, Agilent, Santa Clara, CA, USA) at 3000 cells/well in 100 µL growth medium. Two days after treatment with 3 μM CHIR99021, mitochondrial respiration activity in intact cells was analyzed using a Seahorse Bioscience XFp Extracellular Flux analyzer (Agilent). Briefly, 1 h prior to measuring oxygen consumption, cell culture media was replaced with XF assay medium and maintained in a non-CO₂ incubator for 1 h at 33 °C. Sensor cartridges were placed in the XFp analyzer according to the manufacturer’s instructions from the Mito Stress Test Kit (Agilent, 103010-100). Mitochondrial function was determined by the sequential injection of oligomycin A (1 µM), FCCP (1 µM), and rotenone/antimycin A (0.5 µM). The total protein content in each well was determined after respiration measurement, and all results were normalized to the total protein content.

Mitochondrial respiration activity in HT-22 EV and CHCHD6 KO cells was analyzed using a Seahorse Bioscience XFp Extracellular Flux Analyzer (Agilent). Briefly, cells were seeded in XFp 8-well miniplates (103,025–100, Agilent, Santa Clara, CA, USA) at 8000 cells/well in 100 μL growth medium. One hour prior to measuring oxygen consumption, the cell culture media was replaced with XF assay medium (1 mM pyruvate, 2 mM glutamine, and 10 mM glucose in basal DMEM) and maintained in a non-CO2 incubator for 1 h. The sensor cartridges were placed in the XFp Analyzer according to the manufacturer’s instructions for the Mito Stress Test kit (Agilent, 103,010–100). Mitochondrial function was determined by the sequential injection of oligomycin A (1 μM), FCCP (1 μM), and rotenone/antimycin A (0.5 μM). Following each experiment, the total cellular number of each well was determined using DAPI staining.

3 × 10⁴ C20 wildtype or TSPO knockout cells were grown in XFp 8-well miniplates (Agilent Technologies, Waldbronn, Germany) at 37 °C, humidified air and 5% CO₂. Cartridges were prepared according to the manufacture’s recommendations. The XFp Cell Mito Stress Test Kit (Agilent Technologies, Walsbronn, Germany) contained the mitochondrial stress compounds oligomycin (1 µM), FCCP (2 µM), and rotenone/antimycin A (1 µM). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured by means of a XFp Seahorse Flux Analyzer (Agilent Technologies).