Journal: PLoS Pathogens

Article Title: Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

doi: 10.1371/journal.ppat.1003047

Figure Lengend Snippet: TLR2 −/− , NOD2 −/− , FPR1 −/− and wt mice were inoculated intradermally with S. aureus . (A) Mean total lesion size (cm 2 ) ± SEM. (B) In vivo bioluminescence quantified by mean total flux (photons/s) ± SEM (logarithmic scale). (C) Mean IL-1β protein levels (pg/mg tissue weight) ± SEM from lesional skin at 4 hrs after inoculation. (D) Mean myeloperoxidase (MPO) activity (U/mg tissue weight) ± SEM from lesional skin at 4 hrs after inoculation. * p<0.05, † p<0.01, TLR2 −/− , NOD2 −/− or FPR1 −/− mice versus wt mice (Student's t -test). Data are from 2 experiments with at least 4 mice/group per experiment.

Article Snippet: For all in vitro cultures with mouse neutrophils, neutrophils were obtained from the bone marrow of TLR2-, NOD2, FPR1- and ASC-deficient mice, pIL1-DsRed reporter mice or wt mice by anti-Ly6G MACs magnetic bead separation according to the manufacturer's protocols (Miltenyi Biotec, Inc.).

Techniques: In Vivo, Activity Assay

Journal: PLoS Pathogens

Article Title: Neutrophil-derived IL-1β Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice

doi: 10.1371/journal.ppat.1003047

Figure Lengend Snippet: Neutrophils from mouse bone marrow were infected with live S. aureus (SH1000) or MRSA (USA300 LAC strain) (MOI bacteria∶neutrophils of 5∶1) for a total culture time of 6 hrs and gentamicin was added at 60 min from the start of the infection to prevent bacterial overgrowth. IL-1β protein levels (mean ± SEM) were measured in culture supernatants by ELISA. (A, B) IL-1β protein levels in supernatants from S. aureus -infected neutrophils from TLR2 −/− , NOD2 −/− , FPR1 −/− , ASC −/− and wt mice. (C, D) IL-1β protein levels in supernatants from wt mouse neutrophils infected with S. aureus (C) or MRSA (D), in the absence and presence of an NLRP3-inhibitor (glibenclamide), a caspase-1 inhibitor (Z-YVAD-FMK) or anti-staphylococcal α-toxin antiserum. The respective vehicle controls (DMSO or normal rabbit IgG) for the inhibitors had IL-1β protein levels that did not differ than media alone (data not shown). Data are from 3–5 mice per group. *p<0.05; † p<0.01, ‡ p<0.001, TLR2 −/− , NOD2 −/− , FPR1 −/− or ASC −/− mice versus wt mice (A, B) or the NLRP3-inhibitor, caspase-1 inhibitor or anti-staphylococcal α-toxin antiserum treatment versus media alone (C, D) (Student's t -test).

Article Snippet: For all in vitro cultures with mouse neutrophils, neutrophils were obtained from the bone marrow of TLR2-, NOD2, FPR1- and ASC-deficient mice, pIL1-DsRed reporter mice or wt mice by anti-Ly6G MACs magnetic bead separation according to the manufacturer's protocols (Miltenyi Biotec, Inc.).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Advanced Healthcare Materials

Article Title: A Comparative Inflammation‐on‐a‐Chip with a Complete 3D Interface: Pharmacological Applications in COPD‐Induced Neutrophil Migration

doi: 10.1002/adhm.202301673

Figure Lengend Snippet: A comparative inflammation‐on‐a‐chip model to measure neutrophil transendothelial migration. a) Neutrophil transendothelial migration can be measured in plasma samples from healthy subjects and COPD patients using a single device, therefore enabling comparative analysis. b) A schematic illustration of neutrophil and its representative surface receptors. Neutrophils express the CD11b (integrin alpha M), complement component 5a receptor (C5aR), leukotriene B4 receptor (LTB4R), CXCR1 (IL8RA), CXCR2 (IL8RB), and formyl peptide receptors 1 and 2 (FPR1 and FPR2). The Figure was created using BioRender.com.

Article Snippet: The cells were then stained with fluorescently conjugated primary antibodies, anti‐CXCR1‐APC (Cat. No. 320 612), anti‐CXCR2‐PE‐Cy7 (Cat. No. 320 716), anti‐CD11b‐BV605 (Cat. No. 301 332), anti‐C5aR‐PerCP‐Cy5 (Cat. No. 344 312), anti‐ICAM‐1 (Cat. No. 353 108), anti‐CD62E (Cat. No. 336 012), anti‐CD62P (Cat. No. 304 920) (BioLegend, San Diego, CA, USA), anti‐LTB4R‐FITC (Cat. No. FAB099F), anti‐FPR1‐APC (Cat. No. FAB3744A), or anti‐FPR2‐PE (Cat. No. FAB3744A) (R&D Systems) in FACS buffer containing 2% (v/v) BSA and 2 mM EDTA in DPBS for 30 min at 4 °C.

Techniques: Migration

Journal: Frontiers in Immunology

Article Title: Assessment of Neutrophil Chemotaxis Upon G-CSF Treatment of Healthy Stem Cell Donors and in Allogeneic Transplant Recipients

doi: 10.3389/fimmu.2018.01968

Figure Lengend Snippet: G-CSF treatment affects neutrophil migration. Stem cell donors before and after treatment were analyzed for neutrophil function. (A) Neutrophil count measured before and after G-CSF-treatment in the stem cell donor group ( n = 10). Revealed a significant increase in the number of circulating neutrophils after G-CSF treatment. (B) Distribution of half migration time during chemotaxis within the stem cell donor group before and after G-CSF-treatment. Half migration time was defined as the time required for half-maximal fluorescence signal indicative for migration toward 10 nM fMLP. (C) The distribution of velocity during chemotaxis before and after G-CSF-treatment is indicated. Velocity was measured as Δ %/min at half migration time indicating the speed of the neutrophils when they have reached half maximal fluorescence in the bottom well. Blood cell differentiation analysis (D) before and (E) after purification of neutrophils from stem cell donors before ( n = 3) and after ( n = 4) GCSF treatment confirmed that the purification enriched mature neutrophils >95% independent of previous G-CSF treatment. Statistical analysis was performed using a Wilcoxon signed-rank test (A–C) . * p < 0.05, ** p < 0.01.

Article Snippet: G-CSF treated as well as untreated neutrophils were stained with labeled monoclonal anti-fMLP receptor antibody coupled to PE (PE, MACS, Miltenyi Biotec) in PBS supplemented with 3% FCS in a final dilution of 1:10 and incubated for 30 min at 8°C.

Techniques: Migration, Chemotaxis Assay, Fluorescence, Cell Differentiation, Purification

Journal: Frontiers in Immunology

Article Title: Assessment of Neutrophil Chemotaxis Upon G-CSF Treatment of Healthy Stem Cell Donors and in Allogeneic Transplant Recipients

doi: 10.3389/fimmu.2018.01968

Figure Lengend Snippet: Systemic G-CSF treatment impairs chemotaxis in neutrophils from healthy donors. Purified neutrophils from peripheral blood of healthy, untreated donors ( n = 8) were incubated with 0, 10, or 50 ng/ml human recombinant G-CSF. Thereafter, a chemotaxis assay was performed using fMLP as chemoattractant and half migration time (A) as well as velocity at half migration time (B) was determined. Neutrophils pre-treated with 0, 10, and 50 ng/ml of G-CSF respectively were stained with a PE-labeled anti-fMLP receptor antibody. The histogram is showing a representative experiment of three similar replications (C) . Statistical analysis was performed with a Kruskal-Wallis test for (A) ( p = 0.0098) and (B) ( p = 0.0093), complemented with Mann-Whitney U-tests showing that samples 0 and 50 ng/ml are significantly different for (A,B) with * p < 0.05.

Article Snippet: G-CSF treated as well as untreated neutrophils were stained with labeled monoclonal anti-fMLP receptor antibody coupled to PE (PE, MACS, Miltenyi Biotec) in PBS supplemented with 3% FCS in a final dilution of 1:10 and incubated for 30 min at 8°C.

Techniques: Chemotaxis Assay, Purification, Incubation, Recombinant, Migration, Staining, Labeling, MANN-WHITNEY