Antibodies against human cd68 and tnfα

To promote differentiation into macrophages, isolated human monocytes were cultured in the presence of human recombinant MCSF (20 ng/mL; Peprotech) in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) containing FBS (10%; Life Technologies), l-glutamine (2 mM), and penicillin-streptomycin (Life Technologies) at 37 °C in 5% CO₂. human recombinant MCSF was repeatedly added every 2 days after the initiation of the culture. At day 6, generated macrophages were treated with LPS (10 ng/mL) and incubated for 18 h in the presence or absence of various concentrations of MI-2. The culture supernatants were collected and kept at −80 °C until analysis, and TNFα levels in the culture supernatants were measured using the Human TNFα ELISA MAX^TM Deluxe Kit following the manufacturer’s procedure (BioLegend). To identify human macrophages, cells were stained with antibodies against human CD68 and TNFα (BioLegend) using the BioLegend Intracellular Staining Kit according to the manufacturer’s instructions prior to analysis by flow cytometry. Staining data were analysed on a MACSQuant®VYB flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany).

To induce their differentiation into macrophages, isolated human monocytes were cultured in the presence of human recombinant M-CSF (20 ng/ml; Peprotech, Rocky Hill, NJ, USA) in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) containing FBS (10%; Life Technologies), l-glutamine (2 mM), and penicillin-streptomycin (Life Technologies) at 37°C and 5% CO₂. human recombinant M-CSF was added every 2 days after culture initiation. On day 6, macrophages were treated with LPS (10 ng/ml) and incubated for 18 h in the presence or absence of various concentrations of MI-2. The culture supernatant was collected and maintained at −80°C until analysis, and the TNF-α level in the culture supernatant was measured with the human TNF-α ELISA MAX deluxe kit (BioLegend) according to the manufacturer’s protocol. To identify human macrophages, cells were labeled with antibodies against human CD68 and TNF-α (BioLegend) using the intracellular staining kit (BioLegend) according to the manufacturer’s instructions. Expression data were analyzed on a MACSQuant VYB flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany).