Isolation and Characterization of Mononuclear Phagocytes

Approximately 3 mL whole blood were collected in K2-EDTA blood collection tubes (Rich Science, Chengdu, China) from two healthy male volunteers and patients diagnosed with sepsis who provided informed consent according to the protocol approved by the Scientific and Ethics Committee (IMSS reference number XYFY2021-KL248-01). Peripheral blood mononuclear cells (PBMNCs) were isolated from peripheral blood by Ficoll gradient centrifugation (Amersham Biosciences, Freiburg, Germany). Then, the PBMNCs were suspended in RPMI 1640 cell culture media and incubated for 2 h in 5% (v/v) CO₂ at 37 °C to allow the attachment of mononuclear phagocytes to the bottom plastic surface as previously described 14 (link), 15 (link). The nonadherent cells were removed. The mononuclear phagocytes were washed with prewarm PBS (Kaiji, Nanjing, China) and collected for the indicated experiments. After some of the sample was fixed in 10% neutral buffered formalin for immunofluorescence, the remaining sample was subjected to mitochondrial protein extraction.