Dylight 405 donkey anti rabbit

Isolated human lung cells were stained for SA-β-Gal using the Cell Event™ Senescence Green Flow Cytometry Assay Kit (Invitrogen™, Thermo Fisher Scientific, C10840), following manufacturer’s instructions. Briefly, after dissociation, lung cells were fixed in 2% PFA (Thermo Scientific, 28908) for 10 min at room temperature. Cells were washed, centrifuged, and resuspended in freshly prepared working solution and incubated for 1–2 h at 37 °C in a dry incubator, protected from the light. After incubation, cell suspensions were washed twice with 1% BSA in D-PBS and permeabilized with 0.1% Tween20^® (Sigma, St. Quentin Fallavier, France, P1379) in D-PBS for 15 min on ice. After washing, cells were incubated with anti-LGR6 antibody (1:100; Abcam, Waltham, MA, USA, ab126747) and with APC-conjugated anti-human CD45 and CD31 antibodies (1:1000 and 1:1500; BioLegend, San Diego, CA, USA, 304012 and 303116) for 60 min on ice. Secondary DyLight™ 405 donkey anti-rabbit (1:500; Jackson ImmunoResearch Europe Ltd., 711-475-152) was used to detect LGR6 expression. Samples were run on BD Scientific Canto II Flow Cytometer (BD Bioscience, San Diego, CA, USA, RRID: SCR_018056) and flow cytometry data were analysed with FCS Express™ (version 7, DeNovo Software, Glendale, CA, USA, RRID: SCR_016431).