Fc receptor blocking solution

Live-cell immunofluorescence measurements of cell surface expression for CD11b (integrin ɑ_M), CD18 (integrin β₂) and formyl peptide receptor FPR1 were performed on a Sony SH800 Cell Sorter. All staining and washes were done with cells suspended in phosphate-buffered saline (PBS) containing 2% hiFBS and 0.1% sodium azide, chilled on ice. For each sample, one million cells were first resuspended in 100 μL buffer containing 5 μL Fc Receptor Blocking Solution (Biolegend, #422302) and incubated for 15 minutes. Cells were then spun down and resuspended in 100 μL of buffer containing fluorescently conjugated antibodies for one hour (5 μL of each antibody per sample). Following staining, the samples were washed three times by resuspending in 300 μL of fresh buffer. Following collection of flow cytometry data,.fcs files were exported and processed using the Python package FlowCytometryTools (v. 0.5.1)¹¹⁸ . Gating was performed on the forward and side scatter to isolate the population of live cells (Supplementary Fig. 7).

Human PBMC (~5 x 10⁶ live cells) were stained on ice for 30 min with fluorochrome conjugated rHA probes (180–350 pM) in 100 μL of staining buffer (PBS/2% fetal bovine serum [FBS]) as previously described [8 (link), 30 (link), 54 (link), 78 (link), 79 (link)]. Human PBMC were first treated with Fc receptor blocking solution (BioLegend, Dedham, MA, USA) then stained for 30 min on ice using titrated quantities of fluorescently conjugated monoclonal antibodies (S1 Table). After completion of surface labeling, human PBMC were washed extensively with staining buffer prior to fixation with 1.6% paraformaldehyde in staining buffer for 15 min at RT. Following fixation, cells were pelleted by centrifugation at 400xg for 5 min, resuspended in staining buffer and maintained at 4˚C protected from light until acquisition. Data acquisition was performed using the BD FACSARIA Fusion and analysis performed using FlowJo (FlowJo LLC, Ashland, OR, USA). Compensation values were established prior to acquisition using appropriate single stain controls. PBs were defined as CD3/CD14^neg CD19⁺, CD27⁺, CD38⁺⁺ cells as previously described [67 (link), 80 (link)].

For macrophages staining, macrophages were pretreated with Fc Receptor Blocking Solution (1:20, BioLegend) for 10 min at room temperature and were stained with surface antibodies and Ghost Dye (1:1000, Tonbo Biosciences) at 4°C for 30 min in dark. Macrophages were also fixed and permeabilized with Fixation/Permeabilization Solution (BD Biosciences) and were stained with anti-CD68 (Y1/82A, BioLegend). For T cell intracellular staining, cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer (eBioscience), and intracellular cytokine/ nuclear transcription factor staining was performed according to the manufacturer’s protocol.
The following monoclonal antibodies (mAbs) were used: FITC anti-CD86 (BU63, BioLegend), PE anti-CD163 (GHI/61, BioLegend), APC anti-CD206 (15-2, BioLegend), and PerCP-Cy55 anti-CD68 (Y1/82A, BioLegend), FITC anti-CD4 (A161A1, BioLegend), PE-Cy7 anti-IFNγ (B27, BioLegend), PerCP-Cy55 anti-IL-17A (BL168, BioLegend), Alexa 647 anti-T-bet (O4-46, BD Biosciences), and PE anti-RORγt (AFKJS-9, BD Biosciences). Appropriately matched isotype control mAb to each antigen-specific mAb was used for control.
The stained cells were immediately analyzed on FACSAria II (BD Biosciences) flow cytometer, and data analysis was performed with the FlowJo software (Tree Star).

PBMCs were incubated with Fc receptor blocking solution (BioLegend) and then stained with mixtures of the following monoclonal antibodies against human surface antigens: anti-CD3-Brilliant Violet 421(clone UCHT1), anti-CD4-APC/Fire 750 (clone RPA-T4), anti-CD45RA-Brilliant Violet 605 (clone HI100), anti-CD279 (PD1)-PE(clone EH12.2H7), anti-CD185 (CXCR5)-PE/Dazzle594 (clone J252D4), anti-CD38-FITC (clone T16), anti-HLA-DR-V500 (clone G46-6) for T cell subset analysis; anti-CD19-PE/Dazzle594 (clone HIB19), anti-CD20-Alexa Fluor 700 (clone 2H7), anti-CD27-APC/Fire 750 (clone M-T271), anti-IgD-Brilliant Violet 421 (clone IA6-2), anti-CD180-PE (clone G28-8), anti-CD38-FITC (clone T16), anti-HLA-DR-V500 (clone G46-6), and anti-CD138-APC (clone DL-101) for B cell subset analysis. Following surface staining, cells were washed and stained with 7-AAD Viability Staining Solution. Data were acquired on a FACS LSR Fortessa (BD Biosciences) and analyzed by using FlowJo software (TreeStar).

Lung sections of IPF patients and healthy controls (HCs) were permeabilized with 0.25% Triton X-100 in PBS. After blocking with 10% goat serum and Fc receptor blocking solution (1:25 dilution, BioLegend), lung sections were incubated with anti-Mertk antibody (1:50 dilution, Cell Signaling Technology) overnight at 4 °C, followed by staining with FITC-conjugated secondary antibody (1:500 dilution, Abcam) for 1 h at room temperature. Lung macrophages were labelled with PE anti-human CD68 antibody (1:25 dilution, BioLegend) for 30 min at 4 °C. DAPI was used as a nuclear counterstain. Images of Mertk in CD68⁺ cells were acquired with an Olympus IX83 microscope. The fluorescence intensity of MERTK in CD68⁺ cells was analysed by ImageJ software (NIH, Bethesda, MA, USA).