Lysis buffer

At necropsy time points (day 49 pp for MV/NC and day 5 pc for all other groups), lungs were harvested from all the animals (n = 39) for virological and histopathological evaluation and BALf collection. BALf samples were processed as previously described (30 (link)). Briefly, following the removal of the pluck, the lungs were washed with 50 mL of Ca/Mg-deficient PBS (Corning Life Sciences, Corning, NY, USA). Upon collection, the samples were passed through a 70-µm pore size cell strainer (VWR International, Radnor, PA) to remove mucus plugs, and then centrifuged at 1200 g for 5 min. Subsequently, the BALf was treated with 1× Lysis Buffer (BioLegend, San Diego, CA) for 5 min to lyse RBCs. Following centrifugation, pelleted BALf cells were counted (Countess II Automated Cell Counter, Thermo Fisher Scientific, Carlsbad, CA) and resuspended with the appropriate volume of PBS to achieve a final cell density of 0.5–1 × 10⁵ cells. The cells were stained with a three-step Wright-Giemsa Stain (Quick Stain, VWR International, Radnor, PA) and the BALf cell populations (macrophages, neutrophils, and lymphocytes) were expressed as the percentage of the total number of cells counted (36 (link)). BALf cytology slides were examined by two individuals blinded to treatment groups, including a board-certified veterinary clinical pathologist and the cell counts were averaged.

Blood was collected from mice (50 μL in each staining tube). Each tube received 1 × Lysis Buffer (Biolegend, San Diego, CA, USA) followed by incubation at 37 °C for 10 to 15 min. The cells were centrifuged at 300 × g for 5 min. The supernatant was removed and the cells were stained with peridinin chlorophyll protein complex (PerCP) anti-mouse CD45 antibody, APC anti-mouse CD3 antibody, PE anti-mouse CD8b antibody, FITC anti-mouse CD4 Antibody, True-Nuclear™ One Step Staining Mouse Treg Flow™ Kit (FOXP3 Alexa Fluor® 488/CD25 PE/CD4 PerCP), and FITC anti-mouse IgG1 antibody, PE anti-mouse IgG1 antibody, and APC anti-mouse IgG1 antibody (all from Biolegend, San Diego, CA, USA) as an isotype control. Samples were run on a BD Canto II flow cytometer. The percentage of proliferating T cells was analyzed using FlowJo version 10.

Peripheral blood cells were isolated from residual cells in the leukocyte reduction chamber (TrimaAccel). Cells were eluted by gravity, and RBCs were lysed using 1X lysis buffer (BioLegend). CD34 + progenitor cells were isolated using the CD34 MicroBead UltraPure human KIT (Miltenyi Biotec) and cultured as previously described^{57 (link)}. After 7 wk in culture, cells were maintained IMDM supplemented with 5% FBS, 55 μM 2-ME, 100 ng mL⁻¹ SCF, and 50 ng mL⁻¹ IL-6.