Computational protocol: The Impact of Helicobacter pylori Urease upon Platelets and Consequent Contributions to Inflammation

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Protocol publication

[…] Platelets (5 μL of PRP) kept at 37°C without stirring were stimulated with 100 nM or 300 nM HPU, 25 μg.mL−1 collagen, for 1, 5, and 10 min. Resting platelets (negative controls, no addition) and stimulated platelets in 50 μL of reaction buffer were stained with FITC-labeled anti-CD42 (GP1b) (1:25) (Abcam) and PerCP-labeled anti-CD62P (P-selectin) (1:5) (Abcam) antibodies for 20 min, at room temperature in the dark. After the incubation period, platelets were fixed with 1% paraformaldehyde. Platelets were identified by gating on platelets' size on the basis of forward scatter (FSC) and side scatter (SSC), followed by CD42 expression, a platelet marker. A total of 20,000 events were analyzed for MFI/percentage of CD62P expression. Data were acquired using a FACSCanto II flow cytometer (Becton Dickinson) with BD FACSDiva software and analyzed by Flowjo® vX.In another set of experiments, platelets were previously treated with polyclonal anti-GPVI (1:10) (Santa Cruz Biotech) or monoclonal anti-IIbIIIa (1:10) (Santa Cruz Biotech) for 20 min at room temperature and then stimulated with 100 nM FITC-conjugated HPU for 1, 5, and 10 min at 37°C. Platelets not treated with the antibodies nor exposed to HPU served as negative controls. After the incubation period, cells were fixed with 1% paraformaldehyde. Platelets were identified as described before. A total of 50,000 events were analyzed for percentage of FITC-positive cells. Data were acquired using a FACSCanto II (Beckton Dickinson) with BD FACSDiva software and analyzed by Flowjo® vX. […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Helicobacter pylori, Bacteria
Diseases Blood Platelet Disorders, Exanthema Subitum, Stomach Neoplasms
Chemicals Adenosine Diphosphate, Fluorescein-5-isothiocyanate