Journal: PLoS ONE

Article Title: Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

doi: 10.1371/journal.pone.0116665

Figure Lengend Snippet: Stimulation and platelet-dependent fXII activation.

Article Snippet: The following materials were used: thrombin (Haematologic Technologies; Essex Junction, VT, USA); fXIIa and fXII (Enzyme Research Laboratories; South Bend, IN, USA); AlignFlow flow cytometry alignment beads (2.5 μm for 488-nm excitation), fluorescein-5-isothiocyanate (FITC), and tetramethylrhodamine (TMRM) (Molecular Probes; Eugene, OR, USA); unlabelled and FITC-annexin V (BD Biosciences; San Jose, CA, USA); AlexaFluor-647-annexin V (Biolegend; San Diego, CA, USA); prostaglandin E1 (PGE1) (MP Biochemicals; Irvine, CA, USA); PPACK (EMD Chemicals; Gibbstown, NJ, USA); the chromomeric substrates S2238 and S2302 (Chromogenix; Milano, Italy); HEPES, bovine serum albumin, Sepharose CL-2B, Protein G sepharose, apyrase grade VII, mepacrine (quinacrine), PBS, EDTA and DMSO (Sigma-Aldrich; St Louis, MO, USA); calpeptin and MDL 28170 (Tocris Bioscience; Ellisville, MO, USA); G1/C1-inhibitor and polyclonal goat anti-human serpin G1/C1-inhibitor antibody (R&D Systems; Minneapolis, MN, USA); goat polyclonal anti-human factor XII antibody (LifeSpan BioSciences, Inc., Seattle, WA, USA); peroxidase-AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories; West Grove, PA, USA); egg phosphatidylcholine and egg phosphatidylserine (Avanti Polar Lipids; Alabaster, AL, USA); DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) (AppliChem; Darmstadt, Germany); and a kit to estimate fXII activation (Renam; Moscow, Russia).

Techniques: Activation Assay, Significance Assay

Journal: PLoS ONE

Article Title: Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

doi: 10.1371/journal.pone.0116665

Figure Lengend Snippet: ( A ) We used a scheme that depicts the platelet aggregate to test the C1-INH washout hypothesis. The platelet concentration inside the thrombi was considered 1 per 15 fl. The initial C1-inhibitor distribution in the aggregate for the simulations is shown in grey, and at values ranging from 0.1 to 10 μm/s, the flow penetrated the aggregate. At t = 0, C1-INH at 100 μM (estimated from ref. ) and fXII at 450 nM appeared simultaneously. Factor XII could be activated on the platelet surface, and C1-INH could diffuse through the aggregate (D = 10 μm 2 /s, based on the molecular weight) and move due to the flow. The fXIIa diffusion was assumed negligible because fXII activation is surface-associated ( and this study), and typically, fXIIa is tightly bound to the activation surface . The C1-INH action was described using a mass action equation with the reaction constant 0.00366 μM -1 s -1 . ( B ) Time-course of the distance-averaged [C1-INH] for various flow velocities. ( C ) Time-course of the surface-averaged [fXIIa] for various flow velocities. The C1-INH and fXIIa spatial distributions were governed by a set of differential equations, which were solved using the finite volume solver available within the Virtual Cell environment.

Article Snippet: The following materials were used: thrombin (Haematologic Technologies; Essex Junction, VT, USA); fXIIa and fXII (Enzyme Research Laboratories; South Bend, IN, USA); AlignFlow flow cytometry alignment beads (2.5 μm for 488-nm excitation), fluorescein-5-isothiocyanate (FITC), and tetramethylrhodamine (TMRM) (Molecular Probes; Eugene, OR, USA); unlabelled and FITC-annexin V (BD Biosciences; San Jose, CA, USA); AlexaFluor-647-annexin V (Biolegend; San Diego, CA, USA); prostaglandin E1 (PGE1) (MP Biochemicals; Irvine, CA, USA); PPACK (EMD Chemicals; Gibbstown, NJ, USA); the chromomeric substrates S2238 and S2302 (Chromogenix; Milano, Italy); HEPES, bovine serum albumin, Sepharose CL-2B, Protein G sepharose, apyrase grade VII, mepacrine (quinacrine), PBS, EDTA and DMSO (Sigma-Aldrich; St Louis, MO, USA); calpeptin and MDL 28170 (Tocris Bioscience; Ellisville, MO, USA); G1/C1-inhibitor and polyclonal goat anti-human serpin G1/C1-inhibitor antibody (R&D Systems; Minneapolis, MN, USA); goat polyclonal anti-human factor XII antibody (LifeSpan BioSciences, Inc., Seattle, WA, USA); peroxidase-AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch Laboratories; West Grove, PA, USA); egg phosphatidylcholine and egg phosphatidylserine (Avanti Polar Lipids; Alabaster, AL, USA); DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride) (AppliChem; Darmstadt, Germany); and a kit to estimate fXII activation (Renam; Moscow, Russia).

Techniques: Concentration Assay, Molecular Weight, Diffusion-based Assay, Activation Assay

Journal: Advanced Therapeutics

Article Title: High‐Density Lipoprotein Engineering for Eye‐Drop Treatment of Age‐Related Macular Degeneration

doi: 10.1002/adtp.202300186

Figure Lengend Snippet: Figure 3. CD13 expression in a normal mouse eye and cultured human cells. A) Immunohistochemical analysis of a normal mouse eye. B) Flow cytometry analysis of cultured human corneal epithelial-transformed (HCE-T) cells, human conjunctival cells (Chang conjunctiva), and human umbilical vein endothelial cells (HUVECs) unstained (black) or stained with fluorescein isothiocyanate (FITC)-labeled anti-CD13 antibody (WM15, 5 μL sample−1) (red). The values in the panels indicate the mean fluorescence intensities.

Article Snippet: DC protein assay kit and anti-mouse CD13 monoclonal antibody (R3-63) were purchased from Bio-Rad Laboratories (Hercules, USA).

Techniques: Expressing, Cell Culture, Immunohistochemical staining, Flow Cytometry, Transformation Assay, Staining, Labeling

Journal: Antiviral Research

Article Title: Triggering unfolded protein response by 2-Deoxy- d -glucose inhibits porcine epidemic diarrhea virus propagation

doi: 10.1016/j.antiviral.2014.03.007

Figure Lengend Snippet: 2-DG treatment affects virus packaging. (A) Vero cells were pretreated with 10 mM 2-DG for 24 h, and the cells were analyzed by FACS analysis using anti-CD13 polyclonal antibody. (B, C) 2-DG treatment did not affect virus entry as determined by RT-PCR and Western blot analysis. For RT-PCR analysis, the cells were treated with 10 mM 2-DG before virus infection. Bars represent mean percent copy number of viral RNA levels in cells by RT-PCR using primers specific for PEDV N gene. Cell lysates were analyzed by Western blot using antibody against PEDV N. (D) The Vero cells were infected with PEDV for 2 h (MOI = 1), 2-DG (10 mM) was incubated with the cells. The cells were harvested at different time points. Cell lysates were analyzed by Western blot using antibody against PEDV N. (E, F) The Vero cells was treated with different concentrations 2-DG after infected with PEDV (MOI = 0.01) for 2 h, the ratio of PEDV RNA copy numbers between the supernatants and the cell lysates was detected by Q-PCR, and the ratio of the virus titers in supernatants and cell lysates were determined by plaque assay.

Article Snippet: Detached Vero cells were washed once with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and stained with rabbit anti-human CD13 protein (Boster Bio-Tech Co. Ltd., Wuhan, China) for 1 h at 4 °C.

Techniques: Virus, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Incubation, Plaque Assay