Evaluation of GM-CSF-induced STAT5 Phosphorylation

GM-CSF-stimulated phosphorylation of STAT5 in peripheral blood monocytes (PBMC) and granulocytes was evaluated in six patients according to a modified protocol by Suzuki et al. [11 (link)]. Heparinized blood was processed within 24 hours. Mononuclear cells and granulocytes were isolated using a Ficoll-Paque plus gradient (GE Healthcare, Uppsala, Sweden) and Leucosep tubes (Greiner Bio One, Solingen, Germany). Cell suspensions were incubated with or without GM-CSF (Leukine, Berlex, Bayer Healthcare, Seattle, WA; 100 ng/ml, 15 minutes, 37°C). Expression of phosphorylated STAT5 (pSTAT5) was measured by flow cytometry (BD FACSCanto). In a subset of cases, cell lysates were also evaluated by Western blotting using anti-STAT5 (Santa Cruz Biotechnology; Santa Cruz, CA), anti-phospho-STAT5 (Cell Signaling), or anti-beta actin (Santa Cruz) as primary antibodies.

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Hildebrandt J., Yalcin E., Bresser H.G., Cinel G., Gappa M., Haghighi A., Kiper N., Khalilzadeh S., Reiter K., Sayer J., Schwerk N., Sibbersen A., Van Daele S., Nübling G., Lohse P, & Griese M. (2014). Characterization of CSF2RA mutation related juvenile pulmonary alveolar proteinosis. Orphanet Journal of Rare Diseases, 9, 171.

Publication 2014

Ficoll-Paque plus gradient Leucosep tubes BD FACSCanto anti-STAT5 anti-phospho-STAT5 anti-beta actin

Antibodies Beta actin Blood Cell Ficoll Flow cytometry Gm csf Granulocytes Leukine Patients Peripheral blood monocytes Phosphorylation Stat5

Corresponding Organization :

Other organizations : Ludwig-Maximilians-Universität München, Hacettepe University, Evangelisches Krankenhaus Bielefeld, Boston Children's Hospital, Harvard University, Shahid Beheshti University of Medical Sciences, Newcastle University, Medizinische Hochschule Hannover, Ghent University Hospital

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Variable analysis

independent variables

GM-CSF stimulation (100 ng/ml, 15 minutes, 37°C)

dependent variables

Phosphorylation of STAT5 (pSTAT5) expression in peripheral blood monocytes (PBMC) and granulocytes

control variables

Heparinized blood processed within 24 hours
Mononuclear cells and granulocytes isolated using Ficoll-Paque plus gradient and Leucosep tubes

controls

Cells incubated with and without GM-CSF

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