Journal: Veterinary Research

Article Title: Anti-inflammatory effects of the prostaglandin D 2 /prostaglandin DP1 receptor and lipocalin-type prostaglandin D 2 synthase/prostaglandin D 2 pathways in bacteria-induced bovine endometrial tissue

doi: 10.1186/s13567-022-01100-6

Figure Lengend Snippet: Primer sequences for qPCR

Article Snippet: The following chemicals, reagents, and other materials were used in this study: foetal bovine serum (ExCellBiology, Inc., Shanghai, China); Dulbecco’s modified Eagle medium (DMEM)/F-12, penicillin, and streptomycin (Gibco, Grand Island, NY, USA); amphotericin B (GENERAY, Shanghai, China); bovine interleukin (IL)-6 enzyme-linked immunosorbent assay (ELISA) reagent kit (DY8190) and bovine tumour necrosis factor (TNF)-α duo set (DY2279; R&D Systems, Minneapolis, MN, USA); bovine IL-1β ELISA reagent kit (ESS0027; Kingfisher Biotech, St. Paul, MN, USA); Six-well culture plates (Corning, Inc., Corning, NY, USA); T-PER tissue protein extraction reagent, Halt Protease Inhibitor, Pierce BCA Protein Assay Kit, and prestained protein ladder (Thermo Fisher Scientific, Waltham, MA, USA); sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (TAKARA, Shiga, Japan); centrifugal filter units (Millipore, Billerica, MA, USA); SDS-PAGE kit (Solarbio, Beijing, China); 10X Tris/Glycine buffer (Bio-Rad Laboratories, Hercules, CA, USA); transfer membranes (Millipore); Starting Block T20 (TBS) Blocking Buffer (Thermo Fisher Scientific); Halt Protease Inhibitor (Thermo Fisher Scientific); antibody dilution reagent (Beyotime, Shanghai, China); anti-matrix metalloproteinase (MMP)-2 antibody (Abcam, ab97779, Cambridge, UK); anti-platelet-activating factor receptor (PAFR) antibody (Biorbyt, orb11225, Cambridge, UK); and anti-high mobility group box (HMGB)-1 antibody (Novus Bio, NB100-2322, Littleton, CO, USA); Goat anti-rabbit IgG horseradish peroxidase-linked antibodies and goat anti-mouse IgG horseradish peroxidase-linked antibodies (Cell Signaling Technology, 7074 and 7076, Danvers, MA, USA); goat anti-rabbit IgG H&L antibodies (Alexa Fluor 647) preadsorbed (Abcam, ab150083, Cambridge, UK); AxyPrep Multisource Total mRNA Miniprep Kit (Axygen Scientific, Union City, CA, USA); Primer Script RT Master Mix (Takara); FastStart Universal SYBR Green Master (Rox; Roche, Basel, Switzerland); Luria Bertani broth (Oxoid, Hampshire, UK); Mueller-Hinton II cation adjusted broth (MH broth; BD Biosciences, Franklin Lakes, NJ, USA); and optimal cutting temperature compound (Sakura, Torrance, CA, USA).

Techniques: Sequencing, Concentration Assay

Journal: Veterinary Research

Article Title: Anti-inflammatory effects of the prostaglandin D 2 /prostaglandin DP1 receptor and lipocalin-type prostaglandin D 2 synthase/prostaglandin D 2 pathways in bacteria-induced bovine endometrial tissue

doi: 10.1186/s13567-022-01100-6

Figure Lengend Snippet: PGD 2 -mediated regulation of PAFR and MMP-2 via the L-PGDS/PGD 2 and PGD 2 /DP1 pathways in E. coli - and S. aureus -infected bovine endometrial explants. Immunofluorescence staining, Western blotting, and qPCR results for PAFR ( A ) and MMP-2 ( B ). Data are given as the means ± SEMs. The significance of differences between results was determined by one-way ANOVA, followed by the Dunnett’s test to control for the number of comparisons ( n = 3). Different letters indicate significantly different means ( P < 0.05).

Article Snippet: The following chemicals, reagents, and other materials were used in this study: foetal bovine serum (ExCellBiology, Inc., Shanghai, China); Dulbecco’s modified Eagle medium (DMEM)/F-12, penicillin, and streptomycin (Gibco, Grand Island, NY, USA); amphotericin B (GENERAY, Shanghai, China); bovine interleukin (IL)-6 enzyme-linked immunosorbent assay (ELISA) reagent kit (DY8190) and bovine tumour necrosis factor (TNF)-α duo set (DY2279; R&D Systems, Minneapolis, MN, USA); bovine IL-1β ELISA reagent kit (ESS0027; Kingfisher Biotech, St. Paul, MN, USA); Six-well culture plates (Corning, Inc., Corning, NY, USA); T-PER tissue protein extraction reagent, Halt Protease Inhibitor, Pierce BCA Protein Assay Kit, and prestained protein ladder (Thermo Fisher Scientific, Waltham, MA, USA); sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (TAKARA, Shiga, Japan); centrifugal filter units (Millipore, Billerica, MA, USA); SDS-PAGE kit (Solarbio, Beijing, China); 10X Tris/Glycine buffer (Bio-Rad Laboratories, Hercules, CA, USA); transfer membranes (Millipore); Starting Block T20 (TBS) Blocking Buffer (Thermo Fisher Scientific); Halt Protease Inhibitor (Thermo Fisher Scientific); antibody dilution reagent (Beyotime, Shanghai, China); anti-matrix metalloproteinase (MMP)-2 antibody (Abcam, ab97779, Cambridge, UK); anti-platelet-activating factor receptor (PAFR) antibody (Biorbyt, orb11225, Cambridge, UK); and anti-high mobility group box (HMGB)-1 antibody (Novus Bio, NB100-2322, Littleton, CO, USA); Goat anti-rabbit IgG horseradish peroxidase-linked antibodies and goat anti-mouse IgG horseradish peroxidase-linked antibodies (Cell Signaling Technology, 7074 and 7076, Danvers, MA, USA); goat anti-rabbit IgG H&L antibodies (Alexa Fluor 647) preadsorbed (Abcam, ab150083, Cambridge, UK); AxyPrep Multisource Total mRNA Miniprep Kit (Axygen Scientific, Union City, CA, USA); Primer Script RT Master Mix (Takara); FastStart Universal SYBR Green Master (Rox; Roche, Basel, Switzerland); Luria Bertani broth (Oxoid, Hampshire, UK); Mueller-Hinton II cation adjusted broth (MH broth; BD Biosciences, Franklin Lakes, NJ, USA); and optimal cutting temperature compound (Sakura, Torrance, CA, USA).

Techniques: Infection, Immunofluorescence, Staining, Western Blot, Control

Journal: FEMS Microbiology Letters

Article Title: Fosfomycin suppresses RS-virus-induced Streptococcus pneumoniae and Haemophilus influenzae adhesion to respiratory epithelial cells via the platelet-activating factor receptor

doi: 10.1111/j.1574-6968.2010.02049.x

Figure Lengend Snippet: Suppression by fosfomycin (FOM) of RSV-induced adhesion to A549 cells of FITC-labeled Streptococcus pneumoniae (a) and Haemophilus influenzae (b), as determined by flow cytometry. Two hours before RSV infection, FOM (10 or 100 μg mL −1 ), PAF receptor (PAF-r) antagonist (20 μg mL −1 ), or anti-PAF-r monoclonal antibody (10 μg mL −1 ) was added to cultured A549 cells. The cells were infected with RSV strain Long at MOI 1. After a 24-h infection, FITC-labeled bacterial cells were added to the cell monolayer at MOI 10, and incubation was continued at 37°C for 30 min. The adhered labeled bacteria were detected by flow cytometry (black lines). Gray lines indicate cells incubated with unlabeled bacteria. Each experiment was performed in triplicate. The mean fluorescence intensity was estimated and the relative value to the mean fluorescence intensity of RSV-uninfected cells incubated with FITC-labeled bacteria was calculated. Graph presents the mean of relative fluorescence intensity±SD. * Statistically significant ( P <0.01) from RSV-infected cells without the addition of any inhibitory agents (none).

Article Snippet: A549 cells were harvested from culture flasks using a cell scraper, and then incubated with 2.5 μg mL −1 of mouse anti-PAF receptor monoclonal antibody [11A4 (clone 21); Cayman Chemical, Ann Arbor, MI] or mouse IgG2a,κ isotype control antibody (eBioscience, San Diego, CA).

Techniques: Labeling, Flow Cytometry, Infection, Cell Culture, Incubation, Bacteria, Fluorescence

Journal: PLoS ONE

Article Title: Uptake of oxLDL and IL-10 Production by Macrophages Requires PAFR and CD36 Recruitment into the Same Lipid Rafts

doi: 10.1371/journal.pone.0076893

Figure Lengend Snippet: Macrophages were treated with oxLDL (30 μg/mL) or PAF (10 -7 mol/L) for 20 min at 37°C prior addition of the lysis buffer. Cell lysates were subjected to immunoprecipitating and immunoblotting as described in “Methods section” using antibodies to CD36, PAFR (A) and Flotillin-1 (B). Protein expression was quantified by the AlphaEaseFC™ software V3.2 beta (Alpha Innotech). The autoradiographs show one representative experiment and Graph data are presented as mean ± SEM of four experiments. In A, western blot figures were obtained from the same gel. ** p<0.01 comparing oxLDL-stimulated with the non-stimulated cells (dashed lines).

Article Snippet: The binding specificity was detected using the PAF receptor blocking peptide (Cayman Chemical, Ann Arbor, Michigan, USA) prior to incubation with the PAF receptor IgG antibody.

Techniques: Lysis, Western Blot, Expressing, Software

Journal: PLoS ONE

Article Title: Uptake of oxLDL and IL-10 Production by Macrophages Requires PAFR and CD36 Recruitment into the Same Lipid Rafts

doi: 10.1371/journal.pone.0076893

Figure Lengend Snippet: Macrophages stimulated with oxLDL (30 μg/mL) or PAF (10 -7 mol/L) for 10 min, before staining for PAFR-FITC (green) and CD36-PE (red) and visualized by confocal microcopy (A). Graph data shows the colocalization of PAFR and CD36 in macrophages stimulated with oxLDL for 5, 10 and 20 min (B). Colocalization images were quantified using the package ImageJ 1.44p and Graph data are presented as mean ± SEM of 15 pictures in three independent experiments. ** p<0.01 comparing oxLDL-stimulated with the non-stimulated cells. Images are representative of at least three independent experiments. Yellow patches signify areas of colocalization of CD36 and PAFR.

Article Snippet: The binding specificity was detected using the PAF receptor blocking peptide (Cayman Chemical, Ann Arbor, Michigan, USA) prior to incubation with the PAF receptor IgG antibody.

Techniques: Staining

Journal: PLoS ONE

Article Title: Uptake of oxLDL and IL-10 Production by Macrophages Requires PAFR and CD36 Recruitment into the Same Lipid Rafts

doi: 10.1371/journal.pone.0076893

Figure Lengend Snippet: Frozen sections of human carotid plaques were fixed with acetone and stained with rabbit and with the mouse anti-human CD36 or mouse anti-human CD68. Anti-rabbit IgG DyLight-594 or anti-mouse DyLight-488 were used as a secondary antibody. Colocalization was visualized by confocal microcopy at a 60-fold magnification. In (A), the specificity of anti-PAFR was evaluated by pre-treatment with PAF receptor blocking peptide and after stained for hPAFR. In (B) is shown double staining of PAFR with CD36 or with CD68. Yellow patches signify areas of colocalization. Figures in (A) were used as staining control and were acquired in different magnification.

Article Snippet: The binding specificity was detected using the PAF receptor blocking peptide (Cayman Chemical, Ann Arbor, Michigan, USA) prior to incubation with the PAF receptor IgG antibody.

Techniques: Staining, Blocking Assay, Double Staining, Control

Journal: Clinical & Translational Immunology

Article Title: IgG 3 + B cells are associated with the development of multiple sclerosis

doi: 10.1002/cti2.1133

Figure Lengend Snippet: Antibodies used for human mass cytometry staining

Article Snippet: PAF receptor , 163Dy , AA14 (clone21) , Cayman Chemicals , ✓ , .

Techniques: Mass Cytometry, Membrane