Monocyte Isolation from Sepsis Patients

Blood samples were obtained from six patients with sepsis caused by community-acquired pneumonia within 24 h after admission to the intensive care unit of Amsterdam UMC, location Academic Medical Center (AMC), University of Amsterdam, The Netherlands, and from four healthy subjects (Khan et al., 2020 (link)). Sepsis was defined on the basis of a “probable” or “definite” infection according to the Center for Disease Control and Prevention four-point scale (Garner et al., 1988 (link)) combined with at least one of general, inflammatory, hemodynamic, organ dysfunction, or tissue perfusion parameters derived from the International Sepsis Forum consensus definitions (Calandra et al., 2005 (link)), as previously described in detail (Klouwenberg et al., 2013 (link)). Monocytes were isolated using fluorescence-activated cell sorting (FACS) and stored in RNA protect cell reagent (Qiagen, Hilden, Germany). Total monocytes were identified as CD14⁺CD15⁻ cells in morphologically defined gates and sorted on FACS Canto II flow cytometer (BD Biosciences) with anti-CD14 antibody (Miltenyi Biotech), as previously described (Hoogendijk et al., 2017 (link); Khan et al., 2020 (link)). The Medical Ethics Committee of the Academic Medical Center approved the study (IRB no. 10-056C), and written informed consent was obtained from all patients (or legal representative) and healthy controls.