Gp6^−/− mice show diminished LPS-induced pulmonary inflammation and physiological compromises. (A) Experimental scheme of LPS-induced ALI in C57BL/6J WT and Gp6^−/− mice. LPS was administered at a dose of 5 mg/kg bw intranasally (IN). Sodium chloride (NaCl; 0.9%) was given as vehicle control. Samples were taken 4 hours after administration. (B) Representative micrographs of cytospin preparations of BALF cells stained with DiffQuick staining kit for differential cell counts. Images were acquired using a Thunder Imager DMi8 (Leica Microsystems) equipped with a 20× objective (HC PL APO 20×/0.80 DRY, Leica Microsystems) and a Leica-DMC4500-0265792920 camera (Leica Microsystems). Scale bar, 50 μm. (C) Quantification of total cell count and neutrophilic granulocytes in the BALF using a Neubauer improved hemocytometer and differential cell staining of cytospin preparations. (D) Spectrophotometric determination of MPO activity in the BALF cell pellet (top graph) and whole lung lysates (bottom graph). (E) Quantification of indicated cytokines (pg/mL) and (F) total protein content (μg/mL) in the cell-free BALF via DuoSet enzyme-linked immunosorbent assay kits (R&D Systems) and Pierce BCA Protein Assay Kit (Thermo Scientific), respectively. (G) Pearson correlation between total protein content (μg/mL) in the cell-free BALF and the neutrophil count in the BALF (×10⁴ mL⁻¹) of LPS-treated WT (gray) and Gp6^−/− mice (blue). (H) Quantification of relative hemoglobin (Hb) content in the supernatant after erythrocyte lysis of the BALF cell pellet at 405 nm by spectrophotometry. (I) Determination of change in body temperature, 4 hours after administration of LPS or NaCl. Each data point represents 1 mouse. Bars represent mean ± standard deviation. The two-way analysis of variance (ANOVA) with a Šidák multiple comparisons test. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. TP, thrombocytopenic.