Casy cell counter technology

To determine ATP levels of yeast cells, intracellular metabolites were extracted using hot ethanol. Briefly, 1 × 10⁸ cells were harvested after 42 h culture time at 16,000 g for 2 min at room temperature (RT) and resuspended in 0.5 ml of boiling ethanol (75% ethanol, 10 mM (NH₄)₂SO₄) and incubated in a thermomixer at 1,000 (motor speed) rpm for 3 min at 90°C. Residual cell debris was removed by centrifugation at 16,000 g for 20 min at −5°C, and 10 µl of the supernatant was taken for the subsequent determination of ATP levels using the ATP Determination kit (Invitrogen, Cat. No. A22066). Luminescence was assessed with a microplate reader (GlowMax, PROMEGA, delay time 2 s, integration time 10 s, and detection range 350–650 nm). Data were normalized to the number of cells, as determined by CASY Cell Counter Technology (Schaerfe System, Roche). At least four different clones were measured per strain and construct, each with two technical replicates that were pooled before statistical analysis. This experiment was repeated at least three times independently.

One day after magnetomitotransfer, MRC-5 cells were treated with Accutase (Sigma-Aldrich) and resuspended in 0.6 ml fresh DMEM (Sigma-Aldrich) yielding a cell density of ~2 × 10⁵–4 × 10⁵ cells/ml. Cell densities were determined by CASY Cell Counter technology (Roche Applied Sciences) and used to calculate relative oxygen consumption per cell. 2 ml of cell suspension were applied to high-resolution respirometry assayed at 37 °C with an Oxygraph-2k (Oroboros Instruments) respirometer according to manufactures recommendations and similar to published procedures22 (link). Briefly, after initially recording routine respiration (ROUTINE) the ATP synthase inhibitor oligomycin (2.5 μM) was added, followed by titration with FCCP (0.25 μM steps) leading to an uncoupled state. Finally, complex I and complex III activities were inhibited by rotenone (2.5 μM) and antimycin A (2.5 μM). Magnetomitotransferred cells were always processed in parallel to control cells (i.e. cells treated with microbeads only). Therefore, a paired Student’s t-test (two-sided) was used to detect statistically significant differences between groups after confirming normality and homogeneity of variance.