Hepes ph 7

HBSS with or without calcium and magnesium and glucose was from GE Healthcare Life Sciences (South Logan, UT, USA), Zymosan A from Sigma Aldrich (St. Louis, MO, USA), Ficoll-Paque and Percoll from GE Healthcare Bio-Sciences AB (Uppsala, Sweden), HEPES (pH 7.4) from Sigma. All other used reagents were of research grade. GFP-expressing and chloramphenicol resistant S. aureus (USA300) was a kind gift of Professor William Nauseef (University of Iowa).

Promastigotes of Leishmania (L.) amazonensis (L. amazonensis), strain (MHOM/BR/LTB0016) promastigotes were grown at 25 °C in M199 medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% heat-inactivated fetal calf serum (FCS, Biological Industries, Beit Haemek, Israel), HEPES pH 7.4 (Sigma, St. Louis, MO, USA), 10 mM adenine (Sigma, St. Louis, MO, USA), 4 mM L-glutamine (Biological Industries, Beit Haemek, Israel), 5 μg/mL hemin and 100 U/mL penicillin and streptomycin (Biological Industries, Beit Haemek, Israel). Experiments were performed with logarithmically growing parasites at a cell density of 4–7 × 10⁶ cells/mL.

Peripheral venous blood was collected from subjects into syringes containing 5 U/ml heparin. Mononuclear cells were isolated by differential centrifugation (800 g, 30 min, 20°C) over Lymphoprep and washed twice with sterile phosphate-buffered saline (PBS; GIBCO, Paisley, UK) at 500 g (5 min, 20°C). Cells were resuspended in 10 ml RPMI-1640 medium (Invitrogen) supplemented with 100 U/ml of penicillin (GIBCO), 100 µg/ml streptomycin (GIBCO) and 20 mM HEPES pH 7.4 (Sigma-Aldrich), and plated at a density of approximately 5×10⁶ cells/ml in 8 cm² Nunclon™ Surface tissue culture dishes (Nunc, Roskilde, Denmark) at 37°C, 5% CO₂. After 2 h, non-adherent cells were discarded and 10 ml of fresh RPMI supplemented with 10% foetal bovine serum (FBS; Sigma) added to each tissue culture dish. Cells were then cultured for 5 days at 37°C, 5% CO2, with the addition of a further 10 ml of fresh 10% FBS/RPMI after 24 h. Cells were then washed twice in PBS, scraped and spun down at 500 g (5 min, 20°C). Cells were resuspended into X-vivo-15 medium (Cambrex, MD, USA) and plated at a density of 10⁶ cells per 8 cm² Nunclon™ dish for a further 25 h at 37°C, 5% CO2.

Buffer used in Automated Western Blotting and Western Blotting comprised of 50 mM HEPES pH 7.4 (Sigma, St. Louis, MO, USA), 50 mM sodium fluoride (Sigma, St. Louis, MO, USA), 5 mM sodium pyrophosphate (Sigma, St. Louis, MO, USA), 1 mM ethyldiaminetriacetate (EDTA) (Sigma, St. Louis, MO, USA), 10% v/v glycerol (Sigma, St. Louis, MO, USA), 1% TritonX100 (Sigma, St. Louis, MO, USA) with protease inhibitors benzamidine (Sigma, St. Louis, MO, USA), phenylmethane sulfonyl fluoride (Sigma, St. Louis, MO, USA), and 1 mM dithiothreitol (Sigma, St. Louis, MO, USA).

We obtained whole blood drawn from volunteers at the Stanford Blood Center and prepared enriched B cell fractions using the RosetteSep kit (StemCell Technologies, Cambridge, MA) according to manufacturer’s instructions. We sorted CD19+ IgM+ cells and cultured them at 5 × 10⁵ cells/ml for 5 days at 37 C and 5% CO₂ in RPMI 1640 with L-glutamine (ThermoFisher) supplemented with 10% fetal bovine serum, 10 mM HEPES pH 7.4, 0.1 mM non-essential amino acid (Sigma-Aldrich, St. Louis, MO), 1 mM sodium pyruvate, 100 µ/ml penicillin, 100 µg/ml streptomycin (ThermoFisher), 40 µg/ml apo-transferrin, 500 ng/µl multimeric CD40 ligand (Miltenyi Biotec, San Diego, CA), 200 ng/ml IL-4 (Sigma-Aldrich), and 200 ng/ml IL-10 (Sigma-Aldrich). We extracted RNA from the cells using the RNeasy Micro Kit (Qiagen) according to manufacturer’s instructions, but omitting the DNase digestion step. We then prepared sequencing libraries using 24.5 ng of total RNA as input as described above, except that PCR products were purified using Ampure XP beads at a 0.65:1 ratio instead of a 1:1 ratio before pooling for multiplexed sequencing. We processed the sequences, reconstructed the clonal lineage histories, and measured correlations between the class switch fates of related cells as described above.

Primary cultures of mouse kidney myofibroblasts were isolated from the explantation of 8-day post-ligation renal cortical tissue of C57/BL6 mice, as previously described [56 (link),62 (link)]. Myofibroblasts were grown in DMEM with 4.5 g/L glucose and glutamine (Lonza, Walkersville, MD, USA), 25 mM HEPES pH 7.4 (Sigma-Aldrich, St. Quentin Fallavier, France), 100 µg/mL streptomycin, 100 U/mL penicillin (Pen/Strep), and 10% FCS (Linaris, LaborChemie, Vienna, Austria). Myofibroblasts were pretreated overnight with rhIL-15 (2.5 ng/mL) or the soluble IL-15/IL-15Rα complex composed of rhIL-15 (2.5 ng/mL) precomplexed for 30 min at 37 °C in PBS with 15 ng/mL of rmIL-15Rα-Fc chimeric molecule. Then, myofibroblasts were treated with 2.5 ng/mL of rmTGF-β1 (Bio-Techne Ltd., Lille, France, #7666-MB/CF) for 48 h.