Mitospy green fm

The mitochondrial membrane potential and mitochondrial mass of individual cells were determined using MitoSpy Orange CMTMRos (424803; BioLegend, San Diego, CA, USA), which is useful to indicate cell health and mitochondrial localization, and MitoSpy Green FM (424805, BioLegend), respectively. HEK293 cells (2 × 10⁴/mL) were passaged on glass bottom confocal dishes (30012, SPL), treated with microtubule stabilizers (1 nM Taxol and 2 pM EpD) or a microtubule disturber (10 nM VNB) for 16 h; incubated in DMEM containing 250 nM MitoSpy Green FM and 250 nM MitoSpy Orange CMTMRos for 5 min; and washed with culture medium. Then, nuclei were stained with 1 µg/mL Hoechst 33342^® (H1399, Thermo Fisher Life Technologies) for 5 min at room temperature. Finally, samples were imaged using a confocal microscope with a live-cell chamber system (LSM880, Carl Zeiss AG). For time-lapse live-cell imaging, 120 images were captured at a time interval of 0.5 s. Each image was analyzed and exported as a moving file (25 frames/s) and a single picture (TIFF format) using ZEN2012 software (Carl Zeiss AG). Images were evaluated using ImageJ (version 1.08) with the Difference Tracker plug-in. The movies were used to calculate the average percentage of mitochondrial intensity moving and maximum speed of mitochondria in the cytoplasm.

Culture media α-MEM (α-minimum essential medium), DMEM (Dulbecco’s Modified Eagle’s medium), Medium 199, foetal bovine serum, horse serum, Mitosox, prolong diamond antifade mountant, and mitotracker dyes were all purchased from Thermo Fisher Scientific (UK). α-MEM media-lacking glucose was purchased from PAN Biotech, UK, Mitoquinone (Mito Q) was obtained from Cambridge biosciences, UK). BI605906 was a generous gift from Professor Sir Philip Cohen (MRC Protein Phosphorylation Unit, University of Dundee), but also purchased from Tocris (Bristol, UK), MitoSpy™ Green FM was from BioLegends, UK and MitoPYI, Mitotempo, hepatocyte growth factor, dexamethasone, basic FGF, gelatine, vitamin B12, retinoic acid, VAS2870, palmitate, oligomycin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), rotenone, antimycin-A, hygromycin B, apo-transferrin human, SYBR^® Green JumpStart Taq Ready Mix, and Polyberen were all purchased Sigma-Aldrich, UK).

To determine mitochondrial mass as well as lytic cell death and apoptosis, the cells were analysed by Fluorescence Activated Cell Sorting (FACS). After detaching, both live and dead cells were counted by trypan blue staining in a Neubauer chamber. For subsequent stainings, the cells were resuspended in FACS buffer (1 × PBS + 2% FCS) and cell count was adjusted to 250k cells per well in a v-bottom 96-well plate. For determining cell death, cells were stained for Annexin V and 7AAD according to the manufacturer’s protocol (Biolegend, cat# 640926). Separate samples were stained with MitoSpy Green FM (Biolegend, cat# 424805) to determine the mitochondrial mass. Cells were incubated with MitoSpy dilution (1:10,000 in FACS buffer) for 30 min at 37 °C and subsequently incubated with DAPI solution (1:10,000 in FACS buffer), followed by washing with FACS buffer. Samples were analysed on a FACSFortessa cytometer and FACSDiva software (BD Biosciences).

Isolated, trypsinized cells were incubated with 100 nM TMRM (ThermoFisher) and 500 nM MitoSpy Green FM (BioLegend) at 37°C for 30 min in PBS containing 0.5% BSA and washed with PBS once, followed by FACS analysis.