100 m cell strainer

Lung tissue was surgically removed and freshly transported to our laboratory in RPMI media at 4 °C. The non-involved lung (healthy tissue, HT) and tumor tissue (TT) were separated, minced by scissors and forceps aseptically into small pieces, and enzymatically digested by 250 µg/mL Liberase research-grade (Sigma-Aldrich), 100 µg/mL DNaseI (Sigma-Aldrich) in FCS-free RPMI for 60 min at 25 °C using a magnetic stirrer. Single-cell suspension was filtered through sterile gauze and 100 µm cell strainer (Merck). Following centrifugation (300 g, 5 min), red blood cells were lysed in 5 mL ACK solution (0.15 M NH₄Cl, 10 mM KHCO₃, and 0.1 mM Na₂EDTA at pH 7.3, Molar Chemicals Ltd. Hungary) for 3 min. Following washing (in which 10 mL RPMI was added followed by centrifugation at 300× g for 5 min), cells were counted using a Bürker chamber with viability based on trypan blue dye (Sigma-Aldrich) staining for single-cell mass cytometry.

Mice were killed by cervical dislocation after exposure to CO₂. Kidneys were removed and kept in ice-cold DMEM/F12 medium (Thermo Fisher Scientific, Darmstadt, Germany). The renal capsule was removed under germ-free conditions. Cortex and medulla were separated and chopped into smaller pieces of tissue using a sharp razor blade (Heinz Herenz, Hamburg, Germany). Tissues were incubated in Hanks balanced salt solution/DMEM/F12 (Life Technologies/Gibco^®, Karlsruhe, Germany) containing 1 mg/ml collagenase type 2 (Worthington, Lakewood, USA) for 20 min at 37 °C. The digested tissue was passed through a 100 µm cell strainer (Merck KGaA, Darmstadt, Germany), transferred to a 50 ml falcon tube and washed with ice-cold PBS. After centrifugation at 5100 rpm for 4 min/4 °C, cells were resuspended. After resuspension, the cortical cell pellet was centrifuged at 17500 rpm for 30 min at 4 °C through a 45% Percoll (Ge Healthcare GmbH, Munich, Germany) 55% 2X PBS-Glucose gradient. After washing with ice-cold PBS, tubular preparations were maintained at 37 °C/5% CO₂ in DMEM/F12 supplemented with 1% FBS, 1% Pen/Strep, 1% L-Glutamine (200 mM), 1% ITS (100×), 50 nM hydrocortisone, 5 nM triiodothyronine, and 5 nM Epidermal Growth Factor (Sigma Taufkirchen, Germany). After 24 h, primary cells grew out from isolated tubules.

Splenocytes were prepared from the spleens of 3 weeks old SPF chickens (Rhode Island Red, Roslin) via density gradient centrifugation by using Histopaque 1083 (Merck Life Science) according to the manufacturer’s protocol. Briefly, spleens were mashed through 100 µm cell strainer (Merck Life Science) and suspended in complete Roswell Park Memorial Institute 1640 (RPMI) medium containing 10% FBS and 0.1% penicillin and streptomycin. This was followed by layering with Histopaque 1083 (Merck Life Science) and centrifuging at 400× g for 30 min. The splenocytes were then harvested from the ‘buffy coat’ interface in the density gradient and resuspended in a fresh complete RPMI media. About 2 × 10⁶ cells were plated on each well of 24 well plate and treated with 10 μg of rH9HA or 14 μg of rH9HA-Dec205/CD11c scFv (containing 10 μg rH9HA according to the molecular weight) or 10 μg of scFv or 10 μg of control scFv (anti-H9HA). All cells were stimulated for 5, 22, and 30 h in vitro at 41 °C.