Isolation of Pure CD14++ Monocytes

Monocyte separation was conducted as described previously [19 (link)]. Peripheral blood mononuclear cells were isolated from a 30 ml Lithium-heparin-anti-coagulated blood sample by Ficoll-based density gradient centrifugation. Magnetic cell sorting (autoMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) using CD14 and CD16 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) was performed to deplete CD16⁺⁺ monocytes and to isolate CD14⁺⁺ CD16^—monocytes, followed by flow cytometry after incubation with FITC anti-human CD14 Antibody (BioLegend, San Diego, USA) to ensure purity of the isolated cells.

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Siegler B.H., Uhle F., Lichtenstern C., Arens C., Bartkuhn M., Weigand M.A, & Weiterer S. (2018). Impact of human sepsis on CCCTC-binding factor associated monocyte transcriptional response of Major Histocompatibility Complex II components. PLoS ONE, 13(9), e0204168.

Publication 2018

autoMACS CD14 and CD16 MicroBeads FITC anti-human CD14 Antibody

Anti antibody Blood Density gradient centrifugation Ficoll Fitc Flow cytometry Heparin Human Lithium Microbeads Monocytes Peripheral blood mononuclear cells

Corresponding Organization : University of Giessen

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Variable analysis

independent variables

Magnetic cell sorting (autoMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) using CD14 and CD16 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) to deplete CD16++ monocytes and to isolate CD14++ CD16-- monocytes

dependent variables

Purity of the isolated cells after incubation with FITC anti-human CD14 Antibody (BioLegend, San Diego, USA) measured by flow cytometry

control variables

Peripheral blood mononuclear cells isolated from a 30 ml Lithium-heparin-anti-coagulated blood sample by Ficoll-based density gradient centrifugation

controls

No positive or negative controls were explicitly mentioned.

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