Lsrii fortessa instrument

Six representative SSc-ILD were selected based on having the most available biospecimen in the absence of disease modifying anti-rheumatic drugs (DMARDs). Samples were age/sex matched 1:1 with healthy controls. Cryopreserved PBMCs were thawed in 10 mL of PBS w/o calcium or magnesium, pelleted by centrifugation, resuspended in fluorescence-activated cell sorting (FACS) buffer containing fetal bovine serum and sodium azide and washed once more with FACS buffer. Cells were transferred to a 96-well plate for staining. After incubation with 5 μL Fc Block (BD Biosciences, San Jose, CA) in 45μL FACS buffer for 10 min on ice, surface master mix (Table S2) was added for 30 min on ice without centrifugation prior to adding Live/Dead 700 for an additional 5 min. Cells were pelleted by centrifugation, washed twice with FACS buffer, and resuspended in eBioscience FoxP3 Fix/Perm solution (ThermoFisher Scientific, USA) for 30 min, pelleted by centrifugation, and washed with FoxP3 Fix/Perm buffer. After pelleting by centrifugation, cells were resuspended in intracellular master mix in FoxP3 Fix/Perm buffer for 30 min at RT, pelleted by centrifugation and washed twice with FoxP3 Fix/Perm buffer prior to transferring to FACS tubes for data acquisition. All data were acquired on a BD LSRII Fortessa instrument.

Single cell suspensions of perfused excised lungs were prepared by enzymatic digestion for 45 minutes at 37 °C with Iscove’s Modified Dulbecco’s Medium (IMDM) containing GlutaMax-1 (Gibco, USA) supplemented with 5% fetal bovine serum (FBS; PanBiotech, Germany), 0.2 mg/ml collagenase D (Roche, Germany) and 1 mg/ml DNAse (Sigma-Aldrich, Germany). The enzymatic reaction was stopped with 5 mM EDTA and cell suspensions were filtered through a 100 μm strainer.
Cells were pelleted by centrifugation and resuspended in LIVE/DEAD^® fixable blue stain (ThermoFisher, USA) and anti-mouse CD16/CD32 antibody (purified; BioLegend, USA) as Fc-block following erythrocyte lysis. Cell surface marker staining was performed using anti-mouse CD11b (BioLegend, USA) and anti-mouse F4/80 (BioLegend, USA). All samples were acquired on a BD LSRII Fortessa instrument with FACS DIVA software (BD) and analyzed using FlowJo software (Tree Star). Following debris and dead cell exclusion, AMs were identified as SSChigh, FSChigh, CD11b- and F4/80+ autofluorescent cells. In separate analyses the AM phenotype of the cells identified by this strategy was confirmed by CD11c staining. Absolute cell counts were calculated from the manually counted overall cell number in a sample and the population frequency assessed by flow cytometry.

Whole blood immunophenotyping (WBIP) was carried out on blood samples collected from the macaques enrolled in the ID BCG, aerosol-boosted study, Figure 1c, in order to characterise the γδ T cell profiles in peripheral blood and determine the impact of vaccination. A 50 μL sample of heparinised blood was collected from each animal at each timepoint and incubated for 30 min at room temperature with an antibody cocktail for surface staining, as described above. Erythrocyte contamination was removed using a Cal-lyse reagent kit as per the manufacturer’s instructions (Thermo Fisher Scientific, Swindon, UK). Samples were then fixed in a final concentration of a 4% paraformaldehyde solution (Sigma-Aldrich, Gillingham, UK). Immediately prior to acquisition using an LSRII Fortessa instrument (BD Biosciences, Wokingham, UK), a 50 μL Trucount bead solution (Beckman Coulter, High Wycombe, UK) was added to each sample in order to ascertain the absolute count of each cell population of interest in the blood, as well as their frequencies.

Cells isolated from BAL were pelleted in 1.5 mL microcentrifuge tubes and resuspended in FACS buffer (PBS supplemented with 1% BSA and 0.1% sodium azide). Alternatively, in analyses of PMN infiltration into lung parenchyma, mice received an i.v. injection of anti-CD45 antibody about 10 minutes before sacrifice, and then at time of sacrifice blood was collected, residual blood perfused from lungs with 10 mL of saline, and lungs excised for digest. Fc receptor binding was blocked with 0.25 μg purified anti–mouse CD16/32 antibody specific for FcγRIII/II. Up to 1 × 10⁶ cells were stained for 30 minutes on ice in 50 μL of FACS buffer with the following dyes and antibodies: LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Invitrogen), Ly6C (FITC), Ly6G (APC), CD45.2 (APC-Cy7), MerTK (PE), CD11c (PE-Cy7), CD64 (PerCP-Cy5.5), CD45.1 (Pacific Blue), Siglec-F (BV650), CD115 (BV711). Afterward, cellular events were acquired using a BD LSR-II Fortessa instrument and were analyzed using FlowJo 9.9.6 software.