Anti cd31 apc

Mice were euthanized with avertin overdose and lungs were dissected and examined grossly for tumor formation. Tumors were dissected from the lungs of primary mice and tumor tissue was prepared as described^{2 (link)}. Briefly, tumors were isolated, minced, digested rotating for 1 h at 37 °C with 2 mg/ml collagenase/dispase (Roche), and then filtered twice (100 μm, and then 40 μm) after a 5 min incubation with 0.025 mg/ml DNase. Cell lines were trypsinized and then filtered (40 μm). Single-cell suspensions were stained using anti-CD31-APC (BD Pharmingen 551262), anti-CD45-APC (BD Pharmingen 559864), anti-Epcam-PE-Cy7 (BioLegend 118216), anti-CD24-FITC (BD Pharmingen 553261), anti-Sca-1-APC-Cy7 (BD Pharmingen 560654), with DAPI (Sigma) staining to visualize dead cells. All antibodies were incubated for 15–20 min at 1:100 dilutions. Cell sorting was performed with a Beckman Coulter/Cytomation MoFlo or a BD FACS Aria, and data were analyzed with the FlowJo software (Tree Star Inc.). Example gating strategy is illustrated in Supplementary Figure 9.

Fc regions on cells were blocked with 2 µl Fc-block (BD Pharmingen, 553141) in 50 µl FACS buffer per 10⁶ cells for 10 min on ice. For fibroblast negative selection, the cells were incubated in staining cocktail containing anti-CD31-APC (1 µl/10⁶ cells, BD Biosciences, 561814, Clone: MEC 13.3), anti-CD45-APC (1 µl/10⁶ cells, BD Biosciences, 559864, Clone: 30-F11), anti-CSPG4-AF647 (0.4 µl/10⁶ cells, Bioss, bs-4800R-A647), and anti-CD326-APC (5 µl/10⁶ cells, BD Biosciences, 563478, Clone G8.8) in FACS buffer for 30 min on ice. 4′-6′-diamidino-2-phenylindole (DAPI) was added to the cell suspension before sorting. We gated on DAPI⁻, Epcam⁻, CD31⁻, CD45⁻, NG2⁻ cells after excluding doublets. The cells were sorted using a FACSARIA II (BD Biosciences) into individual wells of 386-well plates containing lysis buffer provided by the Eukaryotic single cell facility (ESCG), SciLifeLabs, (Stockholm, Sweden). For population resorting, cells obtained from MMTV-PyMT mice crossed with the PdgfRα-EGFP reporter mouse^{42 (link)} were stained with anti-CD31-APC, anti-CD45-APC, anti-NG2-AF647, anti-CD326-APC, anti-PDGF-Rβ-biotin (Thermo Scientific, 13-1402-82) for 30 min on ice, followed by 20 min incubation on ice with streptavidin-PE/Cy7 (Thermo Scientific, 25-4317-82).

Dissected corneas were fixed in 2% paraformaldehyde over night at 4 °C. The cornea whole mounts were co-stained with anti CD31- APC (BD Biosciences) and purified rabbit anti mouse LYVE-1 (Abcam) antibodies, followed by secondary goat anti rabbit Alexa Fluor 488 (Invitrogen). The volume of blood and lymphatic vessels was quantified using Volocity version 6.2.1 software (PerkinElmer, Massachusetts, USA). Mouse aortic rings were stained using rat anti-CD31-APC. All Images were captured by Leica SP5-AOBS confocal laser microscope. Mice eyes were snap frozen in OCT (optical cutting temperature compound, Thermo, Waltham, MA, USA). Cryosections were fixed in acetone and immunostained with rat anti-mouse biotinylated CD11b (at dilution 1:100, BD Phamingen), and F4/80 (at dilution 1:100, BD Biosciences) antibodies in conjunction with the HRP conjugated StrepABC kit (Vector Labs, Burlingame, CA) and DAB substrate kit (Vector Labs). Mouse IgG isotype was used as a negative control at dilution 1:100 (BD Biosciences).

Blood was taken from participants before, immediately postexercise and 1 h post exercise bout by a trained phlebotomist using venepuncture at each timepoint. Peripheral blood was drawn into 1 × 6 mL vacutainers spray‐coated with EDTA anti‐coagulant, and 1 × 6 mL serum gel vacutainer (BD Biosciences, UK), with the first 3 mL of peripheral blood discarded. Total blood differential leukocyte counts were determined using an automated hematology analyzer (XS 1000i, Sysmex, UK). Peripheral blood mononuclear cells (PBMC) were isolated using density gradient centrifugation as described elsewhere (Ross et al. 2016). PBMCs were stained with anti‐CD3‐FITC, anti‐CD31‐APC, and anti‐CD28‐PE‐Cy5 antibodies (all BD Biosciences, UK), with subanalysis of T‐cell subsets using anti‐CD4‐PE (Miltenyi‐Biotec, Germany) and anti‐CD8‐PE (BD Biosciences, UK) antibodies to determine CD4⁺ and CD8⁺ T_ANG cells. PBMCs incubated for 30 min prior to enumeration by flow cytometry.
Peripheral blood collected in the serum gel vacutainer was allowed to clot for 30 min prior to centrifugation (1500g for 10 min at 21°C). Serum was obtained and stored at −80°C for analysis of cytomegalovirus (CMV) serostatus.

Prepared single-cell suspensions of digested lung tissue or ex vivo cultured LR-MSC were incubated with fluorescently labeled antibodies for 30 min in the dark situation. Antibodies for identification of MSC characteristics include anti-Sca1-FITC (Miltenyi Biotec, Auburn, CA, USA), anti-CD31-APC, anti-CD45-APC (BD Pharmingen, Franklin Lakes, NJ, USA), anti-CD90.2-FITC (Proteintech, Rosemont, IL), and anti-CD105-FITC (Abcam, ab184667). Expression of cell surface marker of LR-MSC was analyzed with FACSCelesta cytometer (BD Biosciences, San Jose, CA, USA), and data were acquired with BD FACS Diva software. Data were analyzed by FlowJo V10 (Tristar). The LR-MSC in single-cell suspensions of the digested lung was gated by Sca1⁺CD45⁻CD31⁻ and depleted EpCam⁺ epithelial cells using anti-EpCam-PE (Abcam, ab237387). The instilled GFP⁺ LR-MSC was sorted using BD FACSAria II flow cytometry for qPCR analysis.