Sytox blue dead stain

Primary monocytes were cultured and treated as indicated. Thirty minutes before harvest, cells were incubated at 37 °C with 100 nM MitoTracker Green FM probe (Life Technologies) and 100 nM MitoView 633 (Biotium) for 30 m in dark to evaluate mitochondrial mass and ΔΨm, respectively. After harvest, cells were stained with SYTOX Blue dead stain (Life Technologies) as recommened by vendor to monitor cell viability. Data were acquired on a BD LSRFortessa (BD Biosciences). Analysis of data was accomplished as indicated above.

Dermal LECs and BECs were FACS-sorted from the ear skin of 4-OHT–treated 5-wk-old Pik3ca^H1047R;Cdh5-CreER^T2 (n = 5) and Cre⁻ littermate control mice (n = 2) of mixed genders. This stage was chosen as the earliest timepoint showing robust vascular overgrowth, high LEC proliferation and immune cell infiltration in the mutants. One wild-type C57BL/6J mouse, not treated with 4-OHT, was also included, to exclude possible effect of the solvent and 4-OHT on EC transcriptome. Ear skin was first digested in 5 mg/ml Collagenase II in PBS supplemented with 0.2 mg/ml DNaseI and 0.2% FBS, followed by filtering through 50 µm filters (CellTrics, Sysmex). Fc-receptors were blocked with rat anti-mouse CD16/32 antibody (eBioscience), followed by staining using Podoplanin-APC and CD31-Pe-Cy7 antibodies. Dump channel included erythrocytes and immune cells (labeled using CD45-eF450, CD11b-eF450 and Ter119-eF450) and dead cells (SYTOX Blue dead stain; Life Technology). The sorting into 384-well plates was performed with a 100 µm nozzle on BD FACS AriaIII CellSorter (BD BioScience Flow Cytometry System equipped with four lasers: 405, 488, 561, 633 nm). Smart-Seq2 library preparation and sequencing were performed as described previously (Picelli et al., 2014 (link)). Key quality metrics are listed in Table S3.

Cells were stained with anti-human TRAP1-RPE (3H4-2H6, Sigma-Aldrich), primary antibody against HSP90β (EPR16621), STIP1 (EPR6605) and the secondary antibody goat anti-rabbit IgG H&L PE preadsorbed (all Abcam). Mouse IgG1-PE (Invitrogen) and PE-rabbit IgG (Abcam) were used as isotype controls. FcR blocking reagent (Miltenyi Biotec) was used to block non-specific binding. For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained with antibodies for intracellular proteins. For surface and intracellular staining, dead cells were excluded from gating with the use of Sytox Blue dead stain and Fixable Viability Dye eFluor 506 (Invitrogen), respectively.