Ammonium chloride based lysis buffer

Splenocytes from immunized mice were harvested 10 days post-boost (DOL 24) as previously reported (25 (link), 52 (link), 53 (link)) and re-stimulated in vitro to assess cytokine production by flow cytometry. Spleens were mashed through a 70 µM strainer, washed with PBS, and erythrocytes were lysed with 2 min of incubation in ammonium chloride-based lysis buffer (BD Biosciences). Cells were then counted and plated 2 × 10⁶ per well (round bottom 96-well plate) in 200 µl of complete culture medium with or without rHA 10 µg/ml or rHA 10 µg/ml + anti-mouse CD28 2 µg/ml (BioLegend). Plates were incubated for 18 h at 37°C with the addition of Brefeldin A (BD Biosciences) for the last 6 h. Cells were stained against for surface antigens in (PBS + BSA 0.2% + NaN₃ 0.05%) for 20 min at 4°C, then fixed with formalin 2% (10 min at RT) and permeabilized with intracellular staining permeabilization wash buffer (BioLegend) for 20 min at 4°C. Finally, cells were stained with conjugated antibodies against IFNγ, IL- 2, IL-4, and IL-17. Data were acquired on a BD LSRFortessa flow cytometer (BD Biosciences) and analyzed using FlowJo v.10 software (Tree Star). For a complete list of antibodies and fluorochromes used in the study, see Table S1 in Supplementary Material.

Cell suspensions from murine brachial lymph nodes and spleens were generated by mashing through a 70 µM strainer (Thermo Fisher). In spleen suspensions, erythrocytes were lysed by 2 min of incubation in ammonium chloride-based lysis buffer (BD Biosciences). Cells were then counted and plated (1–2) × 10⁶ per well in a flat-bottom 96-well microtiter plate. Cells were incubated with or without recombinant RSV pre-F antigen (1 μg/mL) in 200 μL RPMI 1640 + 10% (v/v) heat-inactivated fetal calf serum (Hyclone), 5 × 10⁻⁶ M β-mercaptoethanol, 1% (v/v) penicillin-streptomycin, 1% (v/v) sodium pyruvate, 1 mM L-glutamine, 100x dilution of non-essential amino acids and 10 mM HEPES (all from Life Sciences). Cells were either incubated for 72 h for measurement of secreted cytokines, or for 24 h in the presence of 0.1% (v/v) BD GolgiPlug (BD Biosciences) for flow cytometry.