Pan monocyte isolation kit 2

Peripheral monocytes were isolated from purified PBMC either with EasySep™ Human Monocyte Enrichment Kit without CD16 Depletion (STEMCELL™ Technologies, Canada) or alternatively with Pan Monocyte Isolation Kit II (Miltenyi Biotec). Monocyte isolation was performed according to the manufacturer's instructions.

After plasma separation, blood cells were diluted 1:2 (v:v) in PBS, and peripheral blood mononuclear cells were enriched by density gradient centrifugation on Pancoll human (Density 1.077 g/mL; PAN Biotech, Aidenbach, Germany). Monocytes were further isolated using the Pan Monocyte Isolation Kit II according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Unlabelled monocytes were resuspended in RPMI 1640 containing 15% fetal calf serum (FCS), 1% nonessential amino acids, 1% Penicillin/Streptomycin, and 1% L-Glutamine and seeded at a density of 1 × 10⁶ cell/mL in Teflon culture bags (Iumox film; Sarstedt, Nümbrecht, Germany). After overnight rest at 37 °C and 7% CO₂, bags were placed on ice for 30 min, and cells were (detached by gently tapping the cooled bags and) transferred to a 50 mL Falcon tube and centrifuged at 300× g for 7 min at 4 °C. Cell pellets were lysed with 350 µL RNA lysis buffer containing β-mercaptoethanol and frozen at −80 °C until RNA isolation and gene expression analysis as described below.

PBMCs were isolated from whole blood, using Lymphoprep gradient media (Axis-Shield, Dundee, UK) and cultured in complete RPMI medium (RPMI medium containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin). PBMCs (2 × 10⁶/ mL) were allowed to settle overnight, prior to experimentation for Figures 1 and 2. NLRP3-inflammasome stimulation was achieved using LPS (10 ng/mL, Ultrapure Escherichia coli K12, Invivogen) for 4 hr, with the addition of ATP (5 mM, Invivogen, San Diego, California) for the final 30 min of stimulation.
Monocytes were isolated by negative selection from PBMCs on the same day using the Pan Monocyte Isolation Kit II (Miltenyi Biotec GmbH), and plated at 1 × 10⁶ cells/mL. All cells were kept in a humidified incubator at 37°C, 5% CO_2.