Roswell park memorial institute (rpmi)

Peripheral blood mononuclear cells were isolated from blood samples by density gradient centrifugation over Ficoll-Paque (Eurobio). Monocytes for macrophage differentiation were isolated by plastic adherence as follows: peripheral blood mononuclear cells were distributed into a 25-cm² flask and allowed to adhere for 1 h at 37°C in RPMI (Gibco) supplemented with 10% FCS (Biowest). Nonadherent cells were removed, and the adherent monocytes were washed twice with medium, detached with Versene (Invitrogen), counted, seeded in 96-well U-bottom plates at a density of 5.10⁴ cells/well, and cultured in RPMI-10% FCS in the presence of 100 ng/ml human GM-CSF (Miltenyi Biotec) in a total volume of 100 µl. At day 6, macrophages were stimulated with 5.10⁵S. aureus, which had been preincubated with 0.5 mg/ml of purified IgA1 or IgA2 and washed. After 24 h, cytokine levels in supernatants were measured using the Cytokine 3-Plex A (IL-6, IL-10, and TNF-α) immunoassay (Quanterix) relying on a Single Molecule Array (Simoa) technology run on a HD-1 Analyzer (Quanterix). Working dilutions were 1:500 in diluent sample. The lower limits of quantification for IL-6, IL-10, and TNF-α were 5.5, 3.65, and 25.5 pg/ml, respectively.

To quantify liver NKT cells following inoculation with PEG-Pam₂Cys, CBA/n mice were injected intravenously with 10 nM PEG-Pam₂Cys or PBS, 72 h prior to liver perfusion into 5 ml RPMI (WAKO Japan) and homogenization with GentleMACS (Miltenyi Biotec, Germany) in 5 ml of RPMI supplemented with 10% FCS and 1% Penicillin. Homogenates were then filtered through a metal mesh into a 50 ml tube with RPMI followed by centrifugation at 1200 rpm for 10 min. Supernatants were discarded and cells were suspended in 25 ml of 33% Percoll (GE Healthcare UK ltd, Buckinghamshire, England) containing 2.5 ml of 5000U/5 ml Heparin (Mochida Pharmaceutical Co. Ltd, Tokyo, Japan) followed by centrifugation at 2200 rpm for 20 min. Red blood cells (RBCs) contained in the pellet were lysed with 3 ml of lysis buffer (0.83% Ammonium Chloride, Tris-HCl buffer pH 7.65) followed by washing with 50 ml HBSS. Cells were suspended in 500  μL RPMI and counted after staining with trypan blue. 50  μL of cell suspension was stained with PE-conjugated anti-mouse (eBioscience) and APC-conjugated anti-mouse DX5 (CD49b) antibodies (Biolegend Inc.) and were analysed by flow cytometry (BD FACSVerse, Nippon Becton Dickinson Company Ltd, Tokyo, Japan).