mouse cd182  (Miltenyi Biotec)

Journal: Clinical and Experimental Immunology

Article Title: Altered neutrophil signalling linked to impaired chemotaxis and increased ROS and NET production in older people with frailty

doi: 10.1093/cei/uxag019

Figure Lengend Snippet: Impaired chemotaxis in frailty neutrophils. (a) Expression of CXCR1 (CD181), CXCR2 (CD182) and CD177 via flow cytometry. (b) A sub-group analysis of FR, HO and HY resulted in significant differences. (c) Chemotaxis of neutrophils towards IL-8, fMLP or random migration (UT). (d) ERK-mediated signalling network predicted to be regulating chemotaxis in FR neutrophils (adj. P = 5.23 × 10 −9 ). Red = up-regulated gene expression, Orange = predicted activation. (e) Representative western blot and densitometry (n = 4) for phosphorylated ERK (ERK-P) in untreated (UT) and 5 min IL-8 treated neutrophils. ERK-P intensity was normalised to Actin. Purple = FR ( n = 4–10), pink = RA ( n = 4–9), orange = HO ( n = 4–10), green = HY ( n = 4–7). Analysed by ANOVA (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Antibodies used were CD177 FITC Monoclonal Antibody (MEM-166, Thermo Fisher, n = 5 per group), CD54 (ICAM-1, n = 7–10 per group) PE-Vio615, REAfinity antibody (Miltenyi, n = 7–10 per group), CD181 (CXCR1) FITC REAfinity antibody (Miltenyi, n = 7–10 per group), CD182 (CXCR2) APC, REAfinity antibody (Miltenyi, n = 7–10 per group), REAfinity isotype controls (APC, PE-Vio615, FITC, Miltenyi), or unstained (US) controls.

Techniques: Chemotaxis Assay, Expressing, Flow Cytometry, Migration, Gene Expression, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Inhibition of IL-27 signaling regulates chemokine levels and sustains CXCR2 receptor expression on mononuclear cells to improve disease outcomes during gram-negative neonatal sepsis

doi: 10.3389/fimmu.2025.1653355

Figure Lengend Snippet: IL-27Rα KO neonatal mice increase CXCR2 chemokine receptor expression during E. coli -induced sepsis. Neonatal WT and KO mice (n=3-4) were subcutaneously inoculated with a target of 2×10 5 CFU/mouse of E. coli O1:K1:H7 or PBS as a control on day 4 of life. Spleens were collected at 24 h post-infection. (A) Heatmap visualization of expression values for a subset of genes annotated as chemokine receptors, interleukins, and chemokine ligands that are differentially expressed between WT and KO spleens in the presence or absence of E. coli infection from our previously published data . Highlighted changes in KO pups (green framed) are contrasted with those in WT pups (red framed). (B) The relative molar concentration (rmc) of a transcript in a RNA sample described previously , was measured and represented in reads per kilobase million values (RPKM). (C) Mean gene expression levels of CXCR2 ± standard error (SE) in the spleen is shown for 3 independent experiments. The expression was determined relative to uninfected control spleens by real-time PCR using the formula 2 -ΔΔCt . Statistical significance in the 95% confidence interval was determined using individual unpaired two tailed t tests; exact P values are shown.

Article Snippet: In addition to Live/Dead stain (FVS780, BD Biosciences, CA, USA), antibodies used for the panel included CD11b (BV786) and Ly6C (PE) from BD Biosciences and CXCR2 (APC) from Miltenyi Biotec.

Techniques: Expressing, Control, Infection, Concentration Assay, Gene Expression, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Inhibition of IL-27 signaling regulates chemokine levels and sustains CXCR2 receptor expression on mononuclear cells to improve disease outcomes during gram-negative neonatal sepsis

doi: 10.3389/fimmu.2025.1653355

Figure Lengend Snippet: CXCR2 expression is selectively increased in splenic mononuclear cells during neonatal infection in the absence of IL-27 signaling. Neonatal WT and KO mice were subcutaneously inoculated with a target inoculum of 2×10 5 CFU/mouse of E. coli O1:K1:H7 (n=5) or PBS (n=5) as a control on day 4 of life. Spleens were collected at 24 h post-infection for enrichment of neutrophils and mononuclear cells. Mean gene expression levels of CXCR2 ± SE in (A) neutrophils or (B) mononuclear cells from a combined 3 independent experiments. The expression was determined relative to uninfected control spleens by real-time PCR using the formula 2 -ΔΔCt . Statistical significance was determined using individual unpaired two tailed t tests; exact P values are shown.

Article Snippet: In addition to Live/Dead stain (FVS780, BD Biosciences, CA, USA), antibodies used for the panel included CD11b (BV786) and Ly6C (PE) from BD Biosciences and CXCR2 (APC) from Miltenyi Biotec.

Techniques: Expressing, Infection, Control, Gene Expression, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Inhibition of IL-27 signaling regulates chemokine levels and sustains CXCR2 receptor expression on mononuclear cells to improve disease outcomes during gram-negative neonatal sepsis

doi: 10.3389/fimmu.2025.1653355

Figure Lengend Snippet: CXCL2 levels regulate CXCR2 receptor expression on neonatal mononuclear cells. Spleens from neonatal WT and KO mice (n=5) were collected on day 7 of life and monocytes were isolated. Cells were stimulated with 0.1 or 1 µg/mL CXCL2 for 8 h Mean gene expression levels of CXCR2 ± SE in (A) WT or (B) KO mononuclear cells are shown for a combined 3 independent experiments. The expression was determined relative to control by real-time PCR using the formula 2 -ΔΔCt . Statistical significance was determined using ANOVA; exact P values are shown. (C) A schematic to illustrate that lower levels of CXCL2 (green) regulate an increase in CXCR2 expression that is turned off by higher levels of ligand (red).

Article Snippet: In addition to Live/Dead stain (FVS780, BD Biosciences, CA, USA), antibodies used for the panel included CD11b (BV786) and Ly6C (PE) from BD Biosciences and CXCR2 (APC) from Miltenyi Biotec.

Techniques: Expressing, Isolation, Gene Expression, Control, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Inhibition of IL-27 signaling regulates chemokine levels and sustains CXCR2 receptor expression on mononuclear cells to improve disease outcomes during gram-negative neonatal sepsis

doi: 10.3389/fimmu.2025.1653355

Figure Lengend Snippet: E. coli -infected IL-27Ra KO neonates have an expanded population of CXCR2 + Ly6C hi mononuclear cells in the spleen. Neonatal WT and KO mice were subcutaneously inoculated with a target of 2×10 5 CFUs of E coli O1:K1:H7 (n=5) or PBS (n=5) as a control on day 4 of life. Spleens were harvested at 24 h post-infection and mononuclear cells isolated by density gradient centrifugation. (A) Cells were then stained with antibodies for CD11b, Ly6C, or CXCR2 and analyzed using flow cytometry. The expression of Ly6C and CXCR2 in the single cell live CD11b + cell gate is shown. (B) The mean percentage ± SE of Ly6C + CXCR2 + cells from a combined 3 independent experiments is shown. Statistical significance was determined using ANOVA; exact P values are shown.

Article Snippet: In addition to Live/Dead stain (FVS780, BD Biosciences, CA, USA), antibodies used for the panel included CD11b (BV786) and Ly6C (PE) from BD Biosciences and CXCR2 (APC) from Miltenyi Biotec.

Techniques: Infection, Control, Isolation, Gradient Centrifugation, Staining, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Inhibition of IL-27 signaling regulates chemokine levels and sustains CXCR2 receptor expression on mononuclear cells to improve disease outcomes during gram-negative neonatal sepsis

doi: 10.3389/fimmu.2025.1653355

Figure Lengend Snippet: Regulation of immune mechanisms during neonatal sepsis in IL-27Rα KO mice. Inhibition of IL-27 signaling in neonatal mice leads to elevated expression levels of CXCR2 in splenic monocytes due to regulated chemokine levels. Upregulation of CXCR2 subsequently enhances the chemotaxis toward CXCL2. The CXCR2 + Ly6C hi mononuclear cells in the spleen promote bacterial clearance and tissue repair in KO neonatal mice during sepsis that ultimately leads to improved survival.

Article Snippet: In addition to Live/Dead stain (FVS780, BD Biosciences, CA, USA), antibodies used for the panel included CD11b (BV786) and Ly6C (PE) from BD Biosciences and CXCR2 (APC) from Miltenyi Biotec.

Techniques: Inhibition, Expressing, Chemotaxis Assay