Zymosan

All mouse experiments were approved by the appropriate institutional and governmental review committees for animal welfare (Thüringer Landesamt für Verbraucherschutz; breeding license 02-052/16). All mouse lines were on a C57BL7/6 background. Primary B cells were isolated from spleens of 8- to 16-week-old mice. Briefly, single cell suspensions were prepared from spleens, depleted of red blood cells using Red Blood Cell Lysis Buffer (Sigma) and purified by negative selection using MACS with anti-CD43 beads (Miltenyi Biotec) according to the manufacturer’s instructions. Isolated B cells were then cultured at 5x10⁵ cells/mL in 24-well plates for 5 days at 37°C in RPMI (Invitrogen) supplemented with 10mM HEPES (ThermoFisher), 50mM β-mercaptoethanol (Sigma), 100U/mL Pen Strep (Gibco) and 10% FBS (Sigma) (cRPMI). Stimulating conditions were achieved by adding 10µg/mL LPS (Sigma, L4391) or 1µg/mL anti-CD40 (eBioscience) with 20ng/mL IL-4 (eBioscience). 3 to 4 hours after seeding, 30µg/mL Zymosan (Wako) or 500µg/mL heat-killed Candida albicans yeast or hyphae were added to the culture. Cultured cells were fed at day 3, maintaining the same culture conditions. After 3 and 5 days of culture, cells were harvested and supernatants stored at -20°C and -80°C for further analysis.

Bupivacaine, capsaicin, and zymosan (Fujifilm Wako Pure Chemical, Osaka, Japan) were dissolved in 5% DMSO and 5% Tween20 in normal saline. Amiodarone (TOA EIYO, Tokyo, Japan) was dissolved in 80°C water and chilled to 20°C, and then diluted with normal saline with 5% DMSO and 5% Tween20. All drugs were freshly prepared before use.

All studies on human blood cells was carried out after an approval from independent ethics committee (Prof. Dr. Ghazala H. Rizwani Chair, IEC, Prof. Qamar Amin, Prof, Dr. Muddasir Uddin, Dr. Shahnaz Ghazi, Dr. Samiuz Zaman, Prof. Dr. Ahsana Dar Farooq, Dr. M. Raza Shah member IEC), International Center for Chemical and Biological Sciences, University of Karachi, No: ICCBS/IEC-008-BC-2015/Protocol/1.0. Informed consents were obtained from the volunteers before drawing the blood. All the experiments were performed in accordance with relevant guidelines and regulations. The approval of institutional ethical committee approval can be seen from supporting information.
LSM (50494, MP Biomedicals, lllkirch, France); HBSS++ and HBSS—(14025, Gibco, California, USA); Ethanol (1070172511, Merck, Darmstadt, Germany); FBS (A11-104, PAA, Pasching, Austria); RPMI-1640 (R8758, Sigma Aldrich, St. Louis, USA); 10 mL sterile syringes (305559, BD Biosciences, NJ, USA); Luminol (A14597, Alfa Aesar, Karlsrushe, Germany); 96-well white half area plates (3693,Costar, NY, USA); Zymosan (26701494, Wako, Shanghai, China).