Apc anti mouse ly 6g ab clone 1a8

Sepsis was induced in C57BL/6 male WT and CIRP^−/− mice by CLP. LDN of WT and CIRP^−/− sham and CLP mice were isolated from 1 ml blood. LDN were stained with APC anti-mouse Ly-6G Ab (clone 1A8; Biolegend) and PerCP/Cyanine5.5 anti-mouse/human CD11b antibody (clone: M1/70; Biolegend) and then accessed by flow cytometry. The population of Ly6G⁺CD11b^hi and Ly6G⁺CD11b^lo were assessed with frequency and real number. The real number was calculated by using Precision Count Beads™ (Catalog no.: 424902; BioLegend). More than 30,000 events were acquired using a BD LSR Fortessa Flow Cytometry Analyzer (BD Biosciences) and the data were analyzed by FlowJo software (Tree Star, Ashland, OR).

Murine bone marrow-derived neutrophil(s) (BMDN) were isolated as described previously (20 (link)). Mice were anesthetized by 2% isoflurane inhalation and the femurs and tibias were dissected. Marrow contents form bones were flushed out with Ca⁺⁺ and Mg⁺⁺ free HBSS using a 25 gauge needle into a petri dish. Suspensions of cells were filtered through a Corning sterile 70 μm cell strainer (Fisher Scientific, Waltham, MA) and BMDN were purified by negative selection using the EasySep mouse neutrophil enrichment kit (Cat. No.: 19762; STEMCELL Technologies, Vancouver, Canada). The purity of the sorted neutrophils was assessed by staining the cells with APC anti-mouse Ly6G Ab (clone 1A8; Biolegend) using BD LSR Fortessa flow cytometer (BD Biosciences). A total of 5 × 10⁵ BMDN were treated with rmCIRP (1 μg/ml) for 4 h. Supernatants were collected and concentrated by trichloroacetic acid (TCA) method as described previously (26 (link)). Protein samples were subjected to Western blot for the detection of NE in the concentrated culture supernatants using anti-mouse NE Ab (Cat. No: MAB 4517-SP; R&D Systems). For normalization blots were stained with Ponceu Red dye.

A number of 2×10⁶ BMDN isolated from WT and TLR4^−/− mice in RPMI 1640 medium were stimulated with PBS or rmCIRP (1 μg/ml). After 4 h 37 °C incubation, the cells were washed and resuspended with 1 ml of 0.5% BSA in PBS. LDN was isolated using the Histopaque gradient as described above. LDN were stained with APC anti-mouse Ly-6G Ab (clone 1A8; Biolegend) and PerCP/Cyanine5.5 anti-mouse/human CD11b antibody (clone: M1/70; Biolegend), and accessed by flow cytometry. The frequency of Ly6G⁺CD11b^hi and Ly6G⁺CD11b^lo were assessed as described above. More than 30,000 events were acquired using a BD LSR Fortessa Flow Cytometry Analyzer (BD Biosciences) and the data were analyzed by FlowJo software (Tree Star).