Computational protocol: Beta2-Adrenoceptor Stimulation Suppresses TLR9-Dependent IFNA1 Secretion in Human Peripheral Blood Mononuclear Cells

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Protocol publication

[…] RPMI 1640 was purchased from Life Technologies (CA, USA) and was supplemented with 10% heat-inactivated fetal bovine serum (FBS Superior, Biochrom AG, Berlin, Germany). Biocoll Separating Solution (1.077g/ml) was also acquired from Biochrom AG. 24- and 96-well flat-bottom plates were obtained from Greiner Bio-One (Frickenhausen, Germany), 24- Transwell Permeable Support Plates (3413, 0.4 µm pore size, polycarbonate membrane, 6.5 mm insert diameter) were purchased from Corning Incorporated Life Sciences (MA, USA). Epinephrine-HCl (E4642), Propranolol-HCl (P0884), Metoprolol-Tartrate (M5391), ICI118,551-HCl (I127), Urapidil-HCl (U100) and RX821002-HCl (R9525) were provided by Sigma-Aldrich (MO, USA) and dissolved in sterile water. CpG ODN 2336 (tlrl-2336) was purchased from InvivoGen (CA, USA), lipopolysaccharide (LPS) was from Sigma-Aldrich (L4130).Reagents for Magnetic Activated Cell Sorting (MACS) and Fluorescent Activated Cell Sorting (FACS) (CD304 Microbead Kit, mouse anti-human-CD14-PerCP antibody, mouse anti-human-CD303-PE antibody, mouse anti-human-CD304-APC antibody and FcR blocking reagent) were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany) and BD Biosciences (CA, USA) (mouse anti-human-CD123-Brilliant-Violet421 antibody), respectively. Additional antibodies were purchased from Abcam (Cambridge, USA): Anti-ADRB2 antibody (polyclonal rabbit, Abcam ab36956), anti-rabbit-IgG antibody (goat, Abcam ab6717) and rabbit IgG-ChIP Grade (isotype control, Abcam ab37415). FACS analysis was performed on a BD FACSCanto II flow cytometer. FACS data was acquired using BD FACSDiva (version 6.1.2) and analyzed using FlowJo (version 10.0.5) software. Assay buffer for FACS and MACS was prepared by adding 0.5% FBS and 2 mM EDTA (0.5 M, pH 8.0, Merck KGaA, Germany) to PBS (pH 7.4, Life Technologies). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Homo sapiens
Diseases Neoplasms, Virus Diseases
Chemicals Epinephrine