Lsr fortessa 2

The antibodies used are displayed in
Table 1.
The lineage cocktail consisted of CD3, CD8α, CD19 and Gr1 (Biolegend, London, UK). Dead cells were excluded using fixable viability dye eFluor 450 (eBioscience, San Diego, CA, USA) (4°C, 15 minutes). Surface staining was carried out in PBS supplemented with 1% FCS (4°C, 15 minutes). Intracellular staining was carried out using Human FoxP3 Buffer (BD Biosciences, Oxford, UK), according to the manufacturer’s instructions. Data were acquired on an LSRFortessa II (BD Biosciences) and analyzed using FlowJo v.X.0.7 (RRID SCR_008520; Tree Star, Ashland, OR, USA).

Flow cytometry assay (using BD LSR Fortessa II) was carried out on hepatic non-parenchymal cells which are composed of the total profile of hepatic leukocyte population. The experiments were performed as published (18 (link)). The following pre-conjugated antibodies were used: CD11B (552850, BD bioscience), CD45 (553083, BD bioscience). Briefly, Hepatic macrophages were defined as viable CD45+ CD11B+ F4/80+ cells from digested livers and used to identify macrophage subsets. Subsets were expressed as proportions of total hepatic macrophages or CD45+ cells. And we collected 10,000 cells every time.

Flow cytometry was performed in adherence to Cossarizza et al. (89 (link)). Flow cytometric Abs are shown in Supplemental Table 2. Cells were extracellularly stained in the presence of the fixable viability dye eFluor 780 (Thermo Fisher Scientific) to exclude dead cells. Staining for intracellular markers was performed using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). Data were acquired using an LSR Fortessa II, A5 Symphony (BD Biosciences), or CytoFLEX (Beckman Coulter) cell analyzer and analyzed using FlowJo, version 10.7.1 (Tree Star).

SH‐SY5Y and ARPE19 cells, stably expressing mcherry‐GFP‐FIS1, were grown on 6‐cm dishes until reaching 70% confluency and treated with either DMSO or PF‐04620110 (5 μM) and PF‐06424439 (5 μM), as indicated, for 24 h in the presence or absence of DFP. Cells were harvested for analysis by washing once with PBS, followed by trypsinisation with trypsin–EDTA (0.25%) (Thermo Fisher Scientific). The cells were then fixed in 3.7% PFA/10 mM Hepes, pH 7.0, for 15 min and finally re‐suspended in 0.4 ml of Dulbecco’s PBS containing 1% FBS into 5‐ml Falcon round‐bottom polystyrene test tubes 12 × 75 mm (Thermo Fisher Scientific).
Flow cytometry data were acquired on an LSR Fortessa II with DIVA software (BD Biosciences). Cells were gated according to their forward‐ and side‐scatter profiles. 488‐nm laser was used to detect GFP in emission filter 530/30 and 561‐nm laser to detect mCherry in emission filter 610/20. Data were analysed using FlowJo software v10.7.1 (BD Biosciences). 20–50,000 cells were analysed per condition, with fluorescent detection in green and red channels. Increased mitophagy was determined for individual cells by detecting decreased green versus red fluorescence, based on gating determined by the green and red fluorescence of vehicle (DMSO)–treated control cells. For treatments, cells were at 60–70% confluency for FACS on the day of the experiment.

For cell surface staining, antibodies conjugated to FITC, PE, APC, Alexafluor 647 (A647), APC-efluor780 (APCe780), and PerCPCy5.5 were obtained from either BD Pharmingen, eBioscience or Biolegend. Fc receptors were blocked using Fc Block (BD Pharmingen). Antibody clones used were: CD4 (RM4-5), CD8a (53-6.7), TCRβ (H57-597), CD44 (IM7), CD45.1 (A20), CD45.2 (104) and CD98 (RC388). Cells were fixed using 1% paraformaldehyde. Standard intracellular cytokine staining protocols were followed for IFNγ (clone XMG1.2; Biolegend) staining. Data were acquired on a LSR Fortessa II with DIVA software or a FACSVerse flow cytometer with FACSuite software (BD Biosciences) and analysed using FlowJo software version 9.9.5 (TreeStar).

The cell cycle analysis was carried out as previously described [29 (link)]. THP-1 cells were treated with ATC cell-derived CM or DMEM (control) for 24 h, after which cells were collected and fixed with cold 70% ethanol and stored at −20 °C for at least 2 h. Then, the cells were washed in PBS buffer and incubated with 1 µg/mL 4′, 6-diamidino-2-phenylindole (DAPI) solution for 10 min before being analyzed. The cell cycle profiles were determined using flow cytometry (LSR Fortessa II, BD Bioscience) and analyzed with FlowJo.