100 μm strainer

Bone marrow was harvested from the femur and tibias of mice [17 (link)]. Harvested cells were resuspended and incubated in ammonium chloride buffer (155 mM NH₄Cl, 0.1 mM Na-EDTA, 10 mM KHCO₃) for 1 min to lyse red blood cells. Cells were centrifuged (1200xg, 5 min) and resuspended in RPMI media and passed through a 100 μm strainer (BD Biosciences; San Jose, CA, USA). The resultant cells were cultured at 1x10⁶ cells/mL in complete medium (RPMI media +10% FCS, 4 mM L-glutamine, 100 U/mL streptomycin/penicillin, 20 mM HEPES pH7.4) supplemented with 20 ng/mL mouse rGM-CSF and 20ng/mL mouse rIL-4. Cultures were maintained at 37^°C under humidified 5% CO₂ conditions for 6 days before experimentation.

Blood and nasal wash were obtained from mice. Briefly, the animals were anesthetized with ether and blood was taken from the abdominal aorta with heparinized syringes. The jaw was incised bilaterally and the nasal cavity, in vicinity to root of the tongue, was exposed. The nasal cavity was washed with saline and the wash was recovered. The blood and the nasal wash were centrifuged at 5,000 rpm for 8 min and stored at −80°C until examination. Then, after thorough washing of the nasal cavity with saline, the upper palates were removed and the NALTs were isolated under the microscope. NALT cells were suspended with 100 μm strainer (BD, Franklin Lakes, NJ) and recovered by centrifugation. The nasal passage cells were prepared as reported [28 (link)]. Briefly, the nasal passage cells were obtained by chipping off from the wall of the nasal cavity, minced, and dispersed with collagenase D (Roche, Basel, Switzerland). The cells were suspended with strainers and separated by biphasic centrifugation with 40% and 75% Percoll (GE Healthcare) at 1,000 rpm for 25 min. The cells in the boundary phase were collected and washed with RPMI 1640 medium.

Villous cytotrophoblast cells (CTBs) were isolated from term placental tissue by enzymatic digestion and Percoll gradient separation, as previously described, with minor modifications (Kallol et al., 2018 (link)). In brief, approximately 50 g of villous tissue was washed in 0.9% NaCl (Sigma–Aldrich) four times for 5 min. Thereafter, the tissue was minced and digested three times with 0.25% trypsin (Sigma–Aldrich) and 300 IU/mL deoxyribonuclease I (Sigma–Aldrich) at 37°C (20 min each). The cell suspension was filtered and overlaid on FBS (Seraglob, Switzerland). After centrifugation at 1,000 × g for 15 min at 10°C, the cell pellet was collected in Dulbecco modified eagle medium (high glucose) basic medium (without FBS) and filtered through 100-μm strainer (BD Biosciences, San Jose, CA, United States). Next, cells were overlaid on a discontinuous Percoll^® (Sigma–Aldrich) density gradient. After centrifugation, CTBs were located at the layer corresponding to 1.046–1.065 g/mL (35–50%) density (Petroff et al., 2006 ). The isolated CTBs were cultured at a density of 1 × 10⁶ cells/cm² in 6-well CellBIND^® plates (Corning, New York, NY, United States) in Dulbecco modified eagle medium (high glucose) supplemented with 10% FBS and 1% antibiotic–antimitotic (Thermo Fisher Scientific). Cells were cultured for 12 h (CTB stage) or 72 h (STB stage).