Human primary monocytes were isolated by layering hyper‐osmotic Percoll solution (48.5% Percoll (Sigma‐Aldrich), 41.5% sterile H2O, 0.16 m filter‐sterilised NaCl) on PBMCs. After 15 min of centrifugation at 580 g, the interphase layer was isolated, and cells were washed with PBS and resuspended in culture medium. To increase the purity of Percoll‐isolated monocytes, the monocytes were adhered to polystyrene flat bottom plates (Corning, Sigma‐Aldrich, New York, USA) or Petri dishes (Falcon, Merck, Darmstadt, Germany) for 1 h at 37°C followed by washing with warm PBS. Next, cells were pre‐incubated with culture medium supplemented with 10% human pooled serum as control, or together with 1 μm BIX‐01294, UNC0638 or NAC. Next, culture medium supplemented with 10% human pooled serum was added as a control, or together with either 2 μg mL−1 β‐glucan, 5 μg mL−1 BCG Intervax, 37.500 microorganisms mL−1 BCG‐Medac, oxLDL (10 μg mL−1), or LPS (1 ng mL−1). After 24 h, cells were washed with warm PBS and culture medium was added. Culture medium was refreshed after 3 days of incubation. On day 6, cells were restimulated with RPMI, LPS (10 ng mL−1), or Pam3Cys (10 μg mL−1). After 24 h, supernatants were collected and stored at −20°C until further use. For RNA sequencing experiments, and trained immunity experiments with CGD patients and healthy controls, monocytes were isolated from PBMCs by negative selection (Pan monocyte isolation kit; Miltenyi Biotech, Bergisch Gladbach, Germany) using a MACS system (Miltenyi Biotech) according to the manufacturer's protocol, to gain a high purity of monocytes.

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