Ic fixation buffer

Lung tissues were minced and enzymatically digested with 1 mg/mL collagenase A (Roche, New York, NY) for 50 min at 37°C. Digested tissue was forced through metal canula and passed through a 75-μm nylon filter to obtain single-cell suspensions. The red blood cells were lysed in lung, lavaged lung and BAL using lysis buffer and cell suspensions were washed with FACS buffer. Cells were re-suspended in FACS buffer and incubated with Fc blocking antibody for 30 minutes, to prevent binding of nonspecific FcγRIII/II. Cells were then labeled with indicated antibodies (as a cocktail: Anti-CD11b, anti-CD11c, anti-CD45 anti-Gr1 anti-Siglec F and anti-MHCII), for 30 minutes on ice. Samples were washed and analyzed using LSR-Fortessa (Beckman Coulter) and data were processed using Flow Jo software (TreeStar, Inc). All antibodies used for flow cytometry were anti-mouse antigens. BrdU 75mg/kg i.p. was administered four hours before the sacrifice of mice. Lung isolation and processed as mentioned above. After incubation with above antibodies cells were fixed with IC fixation buffer (Invitrogen). Fixed cells were permeabilized with permeabilization buffer and incubated for half an hour with anti-BrdU antibody in permeabilization buffer. Finally, cells were washed and processed using Cytoflex flow cytometer (Beckman Coulter).

The RBD-specific stimulation of isolated spleen lymphocytes was performed as described in cytokine secretion detection and positive controls were stimulated eBioscience Cell stimulation cocktail (Invitrogen, USA, #00-4975). After 1 h incubation, 1000 × Brefedin A (Thermo, USA, #00-4506-51) was added to the culture to block cytokine secretion and the samples were incubated for further 4 h. After stimulation, the cells were collected and stained with a cocktail of surface markers antibodies including CD4-APC eFluor780 and CD8a-FITC for 30 min at 4 °C. Following twice washes, the cells were fixed with IC Fixation Buffer (Invitrogen, USA). For 20 min at RT and permeabilized with Cytoperm (Invitrogen, USA). Then the cells were stained with IFNγ-APC, TNF eFluor450, IL-2 Per CP/Cy5.5, IL-4-PE in Cytoperm for 30 min at RT.
All stains were stopped with twice pre-cold PBS washes and the cells were resuspended in appropriate volume PBS, and data were acquired on BD FACSCelesta (BD Biosciences, USA).

Damage to DNA was detected as formation of double-stranded breaks. Cells adhered over a coverslip were fixed with IC Fixation buffer (Invitrogen) for 15 min at room temperature, followed by treatment with 90% alcohol for 7 min. Cells were washed with PBS and incubated with blocking solution containing 0.05% IgG free BSA and 0.05% Triton-X 100 for 30 min. After a wash with PBS, anti-γH2AX antibody was added and incubated for 1 h and 30 min. Cells were washed three times with PBS and incubated with secondary antibody conjugated with Alexa-488. After washing two times with PBS, cells were mounted on ProLong Gold Antifade reagent with DAPI (Invitrogen). Cells were treated with 5 μM cisplatin as positive control for the generation of double-stranded breaks by intercalating DNA strands or with 500 μM CuSO₄ as control for ROS-mediated DNA damage. The formation of γH2AX foci was analyzed under Zeiss AxioObserver Z1 Apotome 2.0 63 × oil NA 1.45. Images were acquired using Zeiss AxioCam MRm Camera and processed using Zeiss Axio vision Rel. 4.8 software. Images were zoomed digitally by Adobe Photoshop CS4.