Anti ly6g apc

Neutrophils or whole bone marrow were stained with the calcium indicator Fluo-4 AM (Life Technologies) at a final concentration of 1μM for 30 min at 37°C, then washed in HBSS−. Whole bone marrow was further stained with anti-Ly6G-APC (eBioscience) for 20 min on ice in order to identify neutrophils specifically. Stained cells were resuspended to a concentration of 2.5 ×10⁶/mL. LY223982 treated neutrophils were incubated for 30 min at 2.5μg/mL prior to analysis by flow cytometry. Immediately prior to collection, stained cells were stimulated with LTB4 (5ng/mL), IL-8 (100ng/mL), HBSS−, or extracted lipid preparations from infected or mock-infected epithelial cells. Lipid preparations were suspended to 400μL of HBSS− then diluted 10-fold in neutrophil suspensions. Lipid stimulated cells were stimulated with at least 3 independent lipid preparations in each condition. Median fluorescent intensity was calculated for gated neutrophils.

Six-week-old wild-type CD45.1⁺ mice (recipient mice) were injected intraperitoneally with Freund’s adjuvant (0.5 ml/mouse) to induce acute peritonitis. Twenty-four hours later, they were injected intravenously with carboxyfluorescein succinimidyl ester (CFSE)-labeled bone marrow cells (10⁷/mouse) from 6-week-old Tipe2^−/− CD45.2⁺ and wild-type CD45.1⁺ mice, mixed at 1:1 ratio. Recipient mice were sacrificed 16 h later, and their peritoneal cells were collected. The peritoneal cells were stained with anti-CD45.1-PE (eBioscience), anti-CD45.2-PerCP/Cy5.5 (eBioscience), and anti-Ly6G-APC (eBioscience), and the percentages of Tipe2^−/−CD45.2⁺CFSE⁺Ly6G⁺ and wild-type CD45.1⁺CFSE⁺Ly6G⁺ cells were determined by flow cytometry. The number of mice required for the experiment was calculated using the Power and Sample Size Calculation (PS) software (Vanderbilt University). The paired Student t-test was used to assess the statistical significance of the results.