Cd19 bv650

To validate results from the GSVA immune enrichment in the LTRC, we used flow cytometry, a non-mRNA related method. Fresh lung tissue samples of IPF patients undergoing bilateral lung transplant at the University of Pittsburgh (USA) were washed with PBS and enzymatically digested as previously described [34 (link)]. Lung homogenates included multiple areas of the same lung lobe, ensuring the representability of the sample to address patient’s heterogeneity. Lung tissue homogenates (10⁶ cells) were then stained 5 min with the viability staining (Fixable viability-Alexa600, BD, USA) and 30 min at 4ºC in the dark with the following conjugated monoclonal antibodies CD3-PECy5.5, CD45-Alexa700, CD16-BV412, CD56-FITC, CD8-V500, CD4-APC-Cy7, CD19-BV650 (BD, USA) and CD14-PE (BioLegend, USA). A minimum of 5 × 10⁵ cells per sample were acquired in a FACS LSRII (BD Biosciences, USA), and data was analyzed using FlowJo v10 (FlowJo LLC, USA). Immune cell populations were determined using the gating strategy depicted in Additional file 1: Fig. S1.

All the cells were incubated with Fc block (Biolegend) for 15 min at room temperature before staining for specific markers. Two million corneal and conjunctival cells were stained for 30 min with CD45 FITC, LY6G APC, CD11b PE (Biolegend). Bone marrow cells were stained with the following panel of antibodies CD3e BUV395, CD19 BV650, c-Kit BV711, CD11b FITC, Ly6G PE, Ter119 PECy7, Ly6C APC, F4/80 APC/Cy7 (BD Biosciences). Appropriate isotypes were used as negative controls. Stained cells were washed with PBS and analyzed on LSR-II flow cytometer (BD Biosciences). The data acquired from LSR-II were analyzed by Summit.

Cell suspensions were stained with violet live/Dead cell stain kit (Life Technologies, Eugene, OR, USA) for the analysis of viability, and then washed and stained with appropriate anti-mouse mAbs CD11b-FITC, F4/80-PECy7, CD11c-PerCP-Cy5.5, CD19-BV650, MHC II (I-A/I-E)-PECy5, Siglec-F-PE, LY6G-APC and LY6C-APC-CY7 (all from eBioscience) for 30 min. For the expression of PD-1, bone marrow–derived macrophages (BMDMs) were collected from cultures and washed with FACS buffer. And then, the cells were stained with PE-conjugated anti-mouse PD-1 (clone J43). For the intracellular staining of macrophages from peritoneal cavity, cells were stained with violet (live/Dead) and membrane markers (CD11b-FITC, F4/80-PECy7, CD11c-PerCP-Cy5.5, CD19-BV650), and then permed with perming buffer (BD Biosciences, San Jose, CA, USA), continued with staining of intracellular cytokines (IL-6-PE and MCP-1-APC, both from eBioscience). Data were acquired on BD fluorescence-activated cell sorting LSR-II (BD Biosciences) and analyzed with Flowjo software (TreeStar, Ashland, OR, USA).