Fluorokine map multiplex

Alveolar macrophages were isolated post mortem via the trachea in 10× 1 mL PBS BAL washes from male or female Pdpn^fl/flVAV1cre⁺ and floxed-only control mice, aged 8–17 weeks, as previously described.11 (link) Cells were cultured for 24 hours either untreated or treated with 0.1 µg/mL LPS in the presence or absence of an α-PDPN (clone 8.1.1) (10 µg/mL). Secreted cytokines and chemokines were measured in the supernatant by Fluorokine MAP Multiplex (R&D Systems, Abingdon, UK). Cells were harvested in ice-cold 5 mM EDTA in PBS using a 25 cm cell lifter for flow cytometry analysis.

Instillations of 40 μg LPS (Escherichia coli O111:B4, InvivoGen, France) in a 50-μl bolus of phosphate-buffered saline (PBS) or PBS alone were administered to each mouse. Mice were euthnaized at 48 h post-LPS instillation (peak of cellular recruitment), or following resolution of the inflammatory response, 9 days post-LPS instillation (28 (link)). Infrared pulse oximetry and pulse distention were assessed by MouseOx Plus (Starr Life Sciences) and bronchoalveolar lavage (BAL) collected as previously described (28 (link)). The epithelial damage marker receptor for advanced glycation end products (RAGE), as well as cytokine and chemokine levels were analyzed using Fluorokine MAP Multiplex (R&D Systems, UK). Whole blood was collected into ethylenediaminetetraacetic acid (EDTA), analyzed on an ABX Pentra 60 (Horbia, UK), and plasma was isolated by centrifugation at 2,000 g for 30 min.

Mice were sacrificed by exsanguination, serum collected and BAL performed with two washes of 0.6 mL PBS/EDTA (2 mM). BAL fluid was centrifuged at 400 g at 4°C with supernatant aliquoted and either used directly or stored at −20°C for analysis of cellular and inflammatory markers. Markers of oedema and endothelial damage—PPI (BioRad protein assay)—epithelial damage—BAL RAGE (DuoSet ELISA, R&D systems, UK)—and inflammation—proinflammatory cytokines interleukin (IL)-6, IL-1β and tumor necrosis factors α (TNFα), neutrophil chemokines CXCL1/KC and macrophage-inflammatory protein-2 (MIP-2), and the epithelial repair growth factor vascular endothelial growth factor (VEGF; Fluorokine MAP Multiplex, R&D systems, UK)—were measured. These parameters were chosen because they are all well-characterised, quantitative markers of damage and inflammation used in several murine models of ALI/ARDS. The remaining cell pellets were analysed directly by flow cytometry.