rme-selectin (cd62e fc chimera  (R&D Systems)

Journal: bioRxiv

Article Title: Cytosolic S100A8/A9 promotes Ca 2+ supply at LFA-1 adhesion clusters during neutrophil recruitment

doi: 10.1101/2024.04.16.589738

Figure Lengend Snippet: (a) ELISA measurements of S100A8/A9 levels in supernatants of WT bone marrow neutrophils stimulated for 10min with PBS, E-selectin or lysed with Triton X-100(mean+SEM, n= 6 mice per group, RM one-way ANOVA, Holm-Sidak’s multiple comparison). (b) Schematic model of the mouse cremaster muscle preparation for intravital microscopy and representative picture of a vessel showing rolling and adherent cells. WT and Mrp14 -/- mice were stimulated i.s. with TNF-α 2h prior to cremaster muscle post-capillary venules imaging by intravital microscopy. Quantification of (c) number or rolling (rolling flux fraction) and (d) number of adherent neutrophils per vessel surface of WT and Mrp14 -/- mice [mean+SEM, n= 5 mice per group, 25 (WT) and 30 ( Mrp14 -/- ) vessels, unpaired Student’s t -test]. (e) Correlation between physiological vessel shear rates and number of adherent neutrophils in WT and Mrp14 -/- mice [ n= 25 (WT) and 30 ( Mrp14 -/- ) vessels of 5 mice per group, Pearson correlation]. (f) Schematic model of sterile inflammation induced by exteriorizing WT and Mrp14 -/- cremaster muscles. (g) Analysis of number of adherent leukocytes by intravital microscopy before and after S100A8/A9 mut intra-arterial injection [mean+SEM, n= 3 mice per group, 3 (WT) and 3 ( Mrp14 -/- ) vessels, 2way ANOVA, Sidak’s multiple comparison]. (h) Representative Giemsa staining micrographs of TNF-α stimulated WT and Mrp14 -/- cremaster muscles (representative micrographs, scale bar =30µm, arrows: transmigrated neutrophils) and (i) quantification of number of perivascular neutrophils [mean+SEM, n= 5 mice per group, 56 (WT) and 55 ( Mrp14 -/- ) vessels, unpaired Student’s t -test]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Briefly, rectangular borosilicate glass capillaries (0.04×0.4mm; VitroCom) were coated with a combination of rmE-selectin (CD62E Fc chimera; 20µg mL −1 ; R&D Systems), rmICAM-1 (ICAM-1 Fc chimera; 15µg mL −1 ; R&D Systems), and rmCXCL1 (15µg mL −1 ; Peprotech) for 3h at RT and blocked with PBS/5% casein (Sigma-Aldrich) over night at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Intravital Microscopy, Imaging, Shear, Sterility, Muscles, Injection, Staining

Journal: bioRxiv

Article Title: Cytosolic S100A8/A9 promotes Ca 2+ supply at LFA-1 adhesion clusters during neutrophil recruitment

doi: 10.1101/2024.04.16.589738

Figure Lengend Snippet: (a) Schematic representation of blood harvesting from WT and Mrp14 -/- mice via a carotid arery catheter and perfusion into self-made flow cambers coated with E-selectin, ICAM-1, and CXCL1. Analysis of (b) number of rolling and (c) number of adherent leukocytes FOV - 1 [mean+SEM, n= 4 mice per group, 10 (WT) and 12 ( Mrp14 -/- ) flow chambers, paired Student’s t -test]. (d) Schematic representation of the soluble ICAM-1 binding assay using bone marrow neutrophils stimulated with PBS control or CXCL1 (10nM) assessed by (e) flow cytometry (MFI=median fluorescence intensity, mean+SEM, n= 5 mice per group, 2way ANOVA, Sidak’s multiple comparison). (f) Spectroscopy fluorescence intensity analysis of percentage of adherent WT and Mrp14 -/- neutrophils, seeded for 5min on ICAM-1 coated plates and stimulated with PBS or CXCL1 (10nM) for 10min (mean+SEM, n= 4 mice per group, 2way ANOVA, Sidak’s multiple comparison). ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Briefly, rectangular borosilicate glass capillaries (0.04×0.4mm; VitroCom) were coated with a combination of rmE-selectin (CD62E Fc chimera; 20µg mL −1 ; R&D Systems), rmICAM-1 (ICAM-1 Fc chimera; 15µg mL −1 ; R&D Systems), and rmCXCL1 (15µg mL −1 ; Peprotech) for 3h at RT and blocked with PBS/5% casein (Sigma-Aldrich) over night at 4°C.

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Comparison, Spectroscopy

Journal: bioRxiv

Article Title: Cytosolic S100A8/A9 promotes Ca 2+ supply at LFA-1 adhesion clusters during neutrophil recruitment

doi: 10.1101/2024.04.16.589738

Figure Lengend Snippet: (a) Representative bright-field pictures of WT and Mrp14 -/- neutrophils spreading over E-selectin, ICAM-1, and CXCL1 coated glass capillaries (scale bar=10μm). Analysis of cell shape parameters (b) area, perimeter, (c) circularity [4π * (Area/Perimeter)] and solidity [Area/Convex area] over time [mean+SEM, n= 103 (WT) and 96 ( Mrp14 -/- ) neutrophils of 4 mice per group, unpaired Student’s t -test]. (d) Rose plot diagrams representative of migratory crawling trajectories of WT and Mrp14 -/- neutrophils in flow chambers coated with E-selectin, ICAM-1, and CXCL1 under flow (2dyne cm -2 ). Analysis of (e) crawling distance, (f) directionality of migration and (g) crawling velocity of WT and Mrp14 -/- neutrophils [mean+SEM, n= 5 mice per group, 113 (WT) and 109 ( Mrp14 -/- ) cells, paired Student’s t -test]. Western blot analysis of ICAM-1 induced (h) Pyk2 and (i) Paxillin phosphorylation of WT and Mrp14 -/- neutrophils upon CXCL1 stimulation (10nM) (mean+SEM, representative western blot of n≥ 4 mice per group, 2way ANOVA, Sidak’s multiple comparison). ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Briefly, rectangular borosilicate glass capillaries (0.04×0.4mm; VitroCom) were coated with a combination of rmE-selectin (CD62E Fc chimera; 20µg mL −1 ; R&D Systems), rmICAM-1 (ICAM-1 Fc chimera; 15µg mL −1 ; R&D Systems), and rmCXCL1 (15µg mL −1 ; Peprotech) for 3h at RT and blocked with PBS/5% casein (Sigma-Aldrich) over night at 4°C.

Techniques: Migration, Western Blot, Comparison

Journal: bioRxiv

Article Title: Cytosolic S100A8/A9 promotes Ca 2+ supply at LFA-1 adhesion clusters during neutrophil recruitment

doi: 10.1101/2024.04.16.589738

Figure Lengend Snippet: (a) Analysis of number of adherent WT and Mrp14 -/- neutrophils under flow as percentage related to the initial number of adherent neutrophils in E-selectin, ICAM-1, and CXCL1 coated flow chambers at indicated shear stress levels. Shear stress was increased every 30sec. [mean+SEM, n= 3 mice per group, 3 (WT) and 3 ( Mrp14 -/- ) flow chambers, unpaired Student’s t -test]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Briefly, rectangular borosilicate glass capillaries (0.04×0.4mm; VitroCom) were coated with a combination of rmE-selectin (CD62E Fc chimera; 20µg mL −1 ; R&D Systems), rmICAM-1 (ICAM-1 Fc chimera; 15µg mL −1 ; R&D Systems), and rmCXCL1 (15µg mL −1 ; Peprotech) for 3h at RT and blocked with PBS/5% casein (Sigma-Aldrich) over night at 4°C.

Techniques: Shear

Journal: bioRxiv

Article Title: Cytosolic S100A8/A9 promotes Ca 2+ supply at LFA-1 adhesion clusters during neutrophil recruitment

doi: 10.1101/2024.04.16.589738

Figure Lengend Snippet: (a) Representative confocal images of LFA-1 staining in WT Lyz2xGCaMP5 and Mrp14 -/- Lyz2xGCaMP5 crawling neutrophils on E-selectin, ICAM-1, and CXCL1 coated flow chambers (scale bar=10μm). (b) Segmentation of LFA-1 signals through automatic thresholding (scale bar=10μm). (c) Size-excluded LFA-1 nanoclusters of 0.15μm 2 minimum size from previously thresholded images (scale bar=10μm). (d) Single cell analysis of average number of LFA-1 nanoclusters in min 0-1, 5-6 and 9-10 of analysis of WT Lyz2xGCaMP5 and Mrp14 -/- Lyz2xGCaMP5 neutrophils [mean+SEM, n= 5 mice per group, 56 (WT) and 54 ( Mrp14 -/- ) neutrophils, 2way ANOVA, Sidak’s multiple comparison]. (e) Representative confocal images of Ca 2+ signals in WT Lyz2xGCaMP5 and Mrp14 -/- Lyz2xGCaMP5 neutrophils (scale bar=10μm) and (f) Ca 2+ signals in the previously segmented LFA-1 nanoclusters (scale bar=10μm). (g) Quantification of subcellular Ca 2+ levels in the LFA-1 nanocluster area in min 0-1, 5-6 and 9-10 in WT Lyz2xGCaMP5 and Mrp14 -/- Lyz2xGCaMP5 neutrophils [mean+SEM, n= 5 mice per group, 56 (WT) and 54 ( Mrp14 -/- ) cells, 2way ANOVA, Sidak’s multiple comparison]. (h) Segmented LFA-1 cluster negative areas (scale bar=10μm) and (i) representative confocal images of Ca 2+ signals in the LFA-1 cluster negative areas (scale bar=10μm). (j) Analysis of cytosolic Ca 2+ levels in the LFA-1 cluster negative areas in min 0-1, 5-6 and 9-10 of WT Lyz2xGCaMP5 and Mrp14 -/- Lyz2xGCaMP5 neutrophils [mean+SEM, n= 5 mice per group, 56 (WT) and 54 ( Mrp14 -/- ) neutrophils, 2way ANOVA, Sidak’s multiple comparison]. (k) Representative confocal micrographs of LFA-1 nanocluster spatial aggregation in WT Lyz2xGCaMP5 and Mrp14 -/- Lyz2xGCaMP5 neutrophils, within 10μm 2 area and minimum 10 LFA-1 nanoclusters considered (≥ 10 LFA-1 nanoclusters within 10µm 2 , yellow circles=spatial aggregation area, scale bar=10μm). (l) Analysis of spatially aggregated LFA-1 nanoclusters of WT Lyz2xGCaMP5 and Mrp14 -/- Lyz2xGCaMP5 neutrophils [mean+SEM, n= 5 mice per group, 56 (WT) and 54 ( Mrp14 -/- ) cells, unpaired Student’s t -test]. (m) Segmentation of WT Lyz2xGCaMP5 and Mrp14 -/- Lyz2xGCaMP5 neutrophil area through Lyz2 channel automatic thresholding and (n) representative confocal images of respective F-actin signals. (o) Analysis of F-actin intensity normalized to the cell area in min 0-1, 5-6 and 9-10 of WT Lyz2xGCaMP5 and Mrp14 -/- Lyz2xGCaMP5 neutrophils [mean+SEM, n= 5 mice per group, 74 (WT) and 66 ( Mrp14 -/- ) cells, 2way ANOVA, Sidak’s multiple comparison]. ns, not significant; *p≤0.05, **p≤0.01, ***p≤0.001.

Article Snippet: Briefly, rectangular borosilicate glass capillaries (0.04×0.4mm; VitroCom) were coated with a combination of rmE-selectin (CD62E Fc chimera; 20µg mL −1 ; R&D Systems), rmICAM-1 (ICAM-1 Fc chimera; 15µg mL −1 ; R&D Systems), and rmCXCL1 (15µg mL −1 ; Peprotech) for 3h at RT and blocked with PBS/5% casein (Sigma-Aldrich) over night at 4°C.

Techniques: Staining, Single-cell Analysis, Comparison