Fix perm kit 1

For each sample, 5×10⁶ cells were used. Cells were stained for viability with Fixable Viability Dye (eBioscience, UK), then Fc receptors were blocked (mouse: anti-CD16/CD32 (clone 9); human: Fc Receptor Binding Inhibitor, both from eBioscience, UK). Cells were surface stained using antibodies listed above, then fixed and permeabilised with the BD Fix/Perm kit I (BD Biosciences). For mouse cells, samples were blocked with 10% goat serum, incubated with rabbit polyclonal anti-LC3 1:400 (Novus Bio, USA), washed, and incubated with Alexa Fluor 488-goat anti-rabbit secondary antibody (Invitrogen, UK). For human cells, samples were incubated with mouse monoclonal anti-LC3-FITC 1:400 (clone 2E6) (Enzo Life Sciences) and mouse monoclonal anti-active capase-3-PE (C92-605, BD Bioscience). Multispectral imaging flow cytometry (MIFC) was performed on an Amnis ImageStream^X instrument. Up to 2.5×10⁵ images were acquired per sample. Cells were gated on aspect ratio to include only singlets, and the gradient root-mean-square feature to include focused cells. Non-viable and apoptotic cells were excluded from analysis based on signal intensity. Using a spot count mask based on the FITC channel of the instrument, the number of LC3⁺ punctae per cell were quantified.

In longitudinal experiments, cytometer settings were standardised using BD Cytometer Setup and Tracking Beads (BD Biosciences). Flow cytometry was performed on BD Canto II or Fortessa instruments. Cell viability was assessed with Live/Dead Green (Invitrogen, UK), or Fixable Viability Dye (eBioscience, UK).
Intracellular staining for p62 was performed following fixation and permeabilisation with BD Fix/Perm kit I (BD Biosciences). In human experiments, mouse monoclonal anti-p62-Alexa Fluor 647 (1 μg/mL) (clone 5F2, MBL, Japan) was used. Mouse cells were incubated with monoclonal rabbit anti-p62 1:500 (clone D10E10) (Cell Signaling Technology, USA), washed, and then incubated with DyLight 649-goat anti-rabbit secondary antibody (Abcam, UK).
For mitochondrial staining, cells were incubated in full media at 37°C for 30 min in the presence of 100 nM Mitotracker Deep Red FM and 100 nM Mitotracker Green (both Invitrogen, UK). Cells were washed and then surface stained.
Cell proliferation was measured by staining cells at room temperature for 10 min with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (eBioscience, UK). Annexin V staining was performed using Annexin V-PE (eBioscience, UK).