Ficoll gradient centrifugation

The study protocol established by Lionetto et al. was used in the experiments [10 (link)]. Ficoll-gradient centrifugation (Amersham Biosciences, Uppsala, Sweden) was used to isolate pooled peripheral blood monocytic cells (PBMCs) from buffy coats of healthy blood donors. RPMI-1640 plus GlutaMAX™ medium (RPMI) (Gibco/Invitrogen, Auckland, New Zealand) supplemented with 5% human serum and 1% antibiotics (10,000 units/mL penicillin G sodium, 10,000 μg/mL streptomycin sulfate, and 25 μg/mL amphotericin B (Gibco standard medium)) was the culture medium for PBMCs at 37°C (humified, 5% CO₂, in 25 cm² tissue culture flasks (Sarstedt)). The nonadherent PBMCs were discarded and the adherent PBMCs were washed twice using 0.1 M phosphate-buffered saline (PBS, pH = 7.2), scraped off, and resuspended in standard medium after 1 h of culturing. These cells were used as monocytic cells (MCs) as described below.
To generate the OC, cell cultures of adherent PBMCs were supplemented with OC differentiation cytokines (10 ng/mL recombinant human macrophage-colony stimulating factor (hM-CSF) and 10 ng/mL recombinant human receptor activator of NF-κB ligand (hRANKL) (ReproTech, Rocky Hill, NJ, USA)).

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.

Ficoll gradient centrifugation (Amersham Biosciences, Freiburg, Germany) was used to isolate peripheral blood mononuclear cells (PBMC) from the peripheral blood of study subjects: Peripheral blood was diluted 1:2 with phosphate buffered saline and gently layered on top of the Ficoll solution. Centrifugation was performed at 20 °C with 400 g for 30 min. Then, the cells in the interphase were aspirated and centrifuged at 20 °C with 300 g for 15 min. The supernatant was discarded and the pellet incubated with erythrocyte lysis buffer for 8 min. After that, the cells were washed two times with phosphate buffered saline and centrifuged (20 °C with 300 g for 10 min). Thereafter, PBMC were prepared and analyzed by flow cytometry.