Facsuite flow cytometry software

Cells were collected, washed, and resuspended in MACS buffer (PBS with 5mM EDTA, 2% (w/v) BSA). Then, cells were stained with anti-PD-L1 antibody recognizing the N-terminus (Abcam ab205921, Cambridge, UK) at 1:200 in MACS buffer or the corresponding rabbit IgG control antibody for 1 h at room temperature. After extensive washing, cells were incubated with donkey-anti rabbit Alexa Fluor^® 488 (no A21206 Thermo Scienfitic, MA, USA) at 1:500 dilution for 1 h at room temperature, followed by one wash step and fixation in 1% (v/v) formaldehyde in MACS buffer. Immuno staining was analyzed by fluorescence flow cytometry on a FACSVerseTM instrument (Becton Dickinson, NJ, USA) using FACSuite Flow Cytometry Software (Becton Dickinson).

Cells were collected, washed, and resuspended in MACS buffer (PBS with 5 mM EDTA, 2% (w/v) BSA). Then, cells were stained with an MCT1-antibody recognizing the N-terminus (Santa Cruz) at 1:200 in MACS buffer or a mouse IgG control antibody (Santa Cruz) for 1 h at room temperature. After extensive washing, cells were incubated with donkey antimouse Alexa Fluor^®-647 (Biolegend, Amsterdam, The Netherlands) at 1:500 dilution for 1 h at room temperature, followed by one wash step and fixation in 1% (v/v) formaldehyde in MACS buffer. Immunostaining was analyzed by fluorescence flow cytometry on a FACSVerseTM instrument (Becton Dickinson, East Rutherford, NJ, USA) using FACSuite Flow Cytometry Software (Becton Dickinson).

The polarization of macrophages was analyzed by flow cytometry. 24 h pi, the lungs were harvested and processed for 30 min at 35 °C and 5% of CO₂, in digestion buffer (1 mg/mL of collagenase A and 0.1 mg/mL of DNase I in Hanks balanced salt solution [HBSS] with 5% fetal bovine serum). Single-cell suspension were stained with a LIVE/DEAD fixable aqua dead cell stain kit (Invitrogen) according to the manufacturer’s instructions. Cells were incubated with a panel of specific antibodies for 30 min on ice in the dark. The panel 3 included I-A/I-E FITC, CD45 PE, CD11c PE-Cy^TM5.5, CD11b PE-Cy^TM7, CD64 APC, CD206 APC-Cy^TM7, and CD80 BV421 (Thermo Fisher Scientific). After staining, cells were washed and fixed with 4% paraformaldehyde. Data were acquired with a BD FACSVerse flow cytometer and analyzed with BD FACSuite flow cytometry software. The gates used to properly interpret the data were determined by fluorescence minus one (FMO) control.