fitc mouse anti rat cd18  (Bio-Rad)

Journal: Nanoscale

Article Title: Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation.

doi: 10.1039/c5nr01851j

Figure Lengend Snippet: Figure 1: Trigger-dependent Microvesicle Shedding. Scanning electron micrograph (a) and size-distribution assessed by NTA (b) of PMN-derived microvesicles originating from PMNs incubated with plasma-opsonized S. aureus bacteria, E. coli, LPS, heat-inactivated bacteria bioparticles or vehicle (HBSS). PMN-derived CD11β/CD18 and CD11β/CD177-double positive events assessed by flow cytometry as a function of bacterial triggering agent (n = 3) (c). Scanning (d,e) and transmission electron micrographs (f,g) of PMNs showing pronounced membrane budding and shedding of microvesicles following incubation with opsonised S. aureus particles for 30 minutes (arrow indicates S. aureus particle) (e,g) compared to PMNs incubated with HBSS (d,f). 3D-tomographies and outer surface reconstructions of PMN incubated with S. aureus further confirmed the constriction of vesicles from the outer membrane seen in TEM (h). Raman spectroscopy maps of PMN incubated with (top, I) or without (bottom, II, control) bacteria showed lipid droplets and peri-membranous accumulation of glycogen granules in stimulated PMNs (I) compared to control (II) (i). PMNs exposed to S. aureus compared to resting PMNs (Figure 1d,e). Transmission electron micrographs of thin sections of PMNs containing phagocytised S. aureus bacteria confirmed increased membrane budding and formation of microvesicles (Figure 1f,g). Formation of glycogen granule clusters, translocation and peri- membranous massing of glycogen granule aggregates, and shipping of cytoplasmatic microvesicles containing glycogen granules were observed in PMNs exposed to bacteria, while glycogen granules remained well-dispersed in the cytoplasm of unstimulated PMNs (Figure 1f,g). 3D-tomography of PMNs further confirmed

Article Snippet: For flow cytometry, FITC mouse anti-rat CD18 (WT3, IgG1, AbD Serotec) and Alexa-647 anti-rat CD11β (OX-42, IgG2a, κ, BioLegend) were used for double staining at a concentration of 1 μg mL−1.

Techniques: Derivative Assay, Incubation, Clinical Proteomics, Bacteria, Flow Cytometry, Transmission Assay, Membrane, Raman Spectroscopy, Control, Translocation Assay, Tomography

Journal: Nanoscale

Article Title: Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation.

doi: 10.1039/c5nr01851j

Figure Lengend Snippet: Figure 3. Microvesicles in Plasma Samples from an Experimental Sepsis Model. Caecal ligation and puncture (CLP) procedure in rats (a). Time-dependent concentration of neutrophil- derived CD11β/CD18-double positive microvesicles assessed by flow cytometry (b). Aggregation of S. aureus bacteria standard with microvesicle isolates from animal plasma at the 24 and 48 hour time point (c) and corresponding ROC curves (d). Characterization of Microvesicle-Bacteria Aggregates In order to better understand the nature of the microvesicle-bacteria aggregates, we used an in vitro analysis to further characterize their properties. The CD11β-positivity of the aggregating human PMN- derived vesicles was confirmed by immunostaining (Figure 4a) and transmission electron micrographs of microvesicle-bacteria aggregates were recorded (Figure 4b). The microvesicle- concentration dependence of bacteria aggregation was confirmed by serially diluting microvesicle isolates from PMNs exposed to S.

Article Snippet: For flow cytometry, FITC mouse anti-rat CD18 (WT3, IgG1, AbD Serotec) and Alexa-647 anti-rat CD11β (OX-42, IgG2a, κ, BioLegend) were used for double staining at a concentration of 1 μg mL−1.

Techniques: Clinical Proteomics, Ligation, Concentration Assay, Derivative Assay, Flow Cytometry, Bacteria, In Vitro, Immunostaining, Transmission Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Protective Effects of Modeled Superoxide Dismutase Coordination Compound (MSODa) Against Ischemia/Reperfusion Injury in Rat Skeletal Muscle.

doi: 10.1159/000430369

Figure Lengend Snippet: Fig. 1. Cytometry evaluation of the positive rate of CD11b/CD18 after ischemia (A), at 12 h after I/R injury (B), I/R model rats treated with SOD (C) or MSODa (D).

Article Snippet: Monoclonal antibodies for ICAM-1 (MCA733), CD11b-RPE (MCA711PE), and CD18-FITC (MCA775F) were purchased from AbD Serotec Bio Comp (Oxford, UK).

Techniques: Cytometry

Journal: Circulation

Article Title: Nicotine strongly activates dendritic cell-mediated adaptive immunity: potential role for progression of atherosclerotic lesions.

doi: 10.1161/01.cir.0000047279.42427.6d

Figure Lengend Snippet: Figure 5. Functional improvement of antigen-presenting cells after stimulation with nicotine (107 mol/L). In MLRs of human DCs mixed with allogeneic T lym- phocytes, nicotine-prestimulated DCs (black bars) induced T-cell activation, which was measured as secretion of T cell–specific cytokine IL-2. Similar activa- tion was obtained by standard doses (100 ng/mL) of LPS (stippled bars) (A). Similar T cell–stimulatory effects of nico- tine could be found for monocytes (MCs) along with allogeneic T cells but to a lesser extent than DCs (B). To also prove stimulating effects of nicotine for antigen- specific assays, we used OVA-peptide– loaded murine DCs in combination with OVA-transgenic T cells (C). To assess T-cell proliferation in response to alloge- neic nicotine-prestimulated human DCs or monocytes, we measured loss of incorporated CSFE labeling by FACS analysis, resulting in cells with lower lev- els of fluorescence of CSFE. To detect T helper cells, we performed a dual stain- ing together with allopyocyanine-labeled CD4 antibodies (D). Nicotine-stimulated DCs significantly enhanced CD40L (CD154) expression on T cells in DC–T- cell cocultures as assessed by CD3- FITC/CD40L-PE double stainings (E). Enhanced T-cell–stimulatory capacity of nicotine-stimulated human DCs is paral- leled by a decrease in internalization of soluble antigens such as FITC-dextran (F). Data are given as meanSEM (n3) and representative FACS analyses out of 3 independent experiments are chosen.

Article Snippet: Then, cells were incubated with mouse anti-human CD11a-FITC, CD18-FITC, CD54-FITC, CD83-FITC, CD86-FITC, HLA-DR-FITC (all from BD Pharmingen), and CD40-FITC (Serotec).

Techniques: Functional Assay, Activation Assay, Transgenic Assay, Labeling, Staining, Expressing