Cd45 percp

Mononuclear cells from the blood, bone marrow, and lung tissue were obtained, as previously described on p189 [20 (link),54 (link)], followed by flow cytometric analysis of the expression of surface markers of mouse mononuclear cells. Briefly, cell suspensions were stained with the following fluorophore-conjugated monoclonal antibodies: CD45 PerCP, CD31 APC, CD34 FITC, CD146 PerCP-Cy5.5, CD309 (Flk-1) APC, and CD117 (c-kit) PeCy7 (all Becton Dickinson, San Jose, CA, USA). Appropriate isotype controls were used. Labeled cells were thoroughly washed with PBS and analyzed on a FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA) using FACS Diva software. At least 100,000 events were recorded for each sample.

T, B, and NK cell counts were determined in months + 1, + 2, + 3, and + 6 post-transplantation. Lymphocyte subsets in peripheral blood were quantified using Trucount Tubes (Becton Dickinson, Albertslund, Denmark) together with the following panel of conjugated monoclonal antibodies and analyzed on a FC500 flow cytometer (Beckman Coulter, Copenhagen, Denmark): CD3-PerCP, CD3-FITC, CD4-FITC, CD8-PE, CD45-PerCP, CD16/56-PE, CD20-FITC, and CD19-PE (Becton Dickinson). CD3⁺ T cells, CD3⁺CD4⁺ T cells, and CD3⁺CD8⁺ T cells were determined. NK cells were differentiated by CD3⁻CD45⁺CD16⁺CD56⁺ phenotype. The following B cell phenotypes were distinguished: total B cells (CD45⁺CD19⁺), mature B cells (CD45⁺CD19⁺CD20⁺), and immature B cells (CD45⁺CD19⁺CD20⁻). Data of these immune cell populations have been published previously in a different context [46 (link)].

Blood was drawn from WD-fed mice in the presence of anti-coagulant (EDTA-2K). After Fc receptor blocking (#553141, BD), cells were stained with fluorescently labelled antibodies F4/80-Alexa488 (#MF48020, 1:20, invitrogen), CD11b-PE (#561689, 1:20, BD) and CD45-PerCP (#561047, 1:20, BD Biosciences) followed by FACS Lysing Solution (#349202, BD Biosciences), according to the instruction manual. Circulating foam cell cluster in high SSC subsets of F4/80⁺CD11b⁺CD45⁺ were measured by Attune Acoustic Focusing Cytometer (Applied biosystems) with modifications as reported previously^{56 (link)}.

Platelet-leukocyte aggregate formation was studied by flow cytometry. In vivo, blood was collected via a tail cut before (0 h), 2 h, and 4h after PLCA. In vitro, washed platelets were prepared from PRP, resuspended in HEPES buffer, and activated with ADP (20μmol/L) at 37°C. Leukocytes were isolated from the sediments obtained after the centrifugation and removal of PRP. After lysis of erythrocytes (Pharmlyse kit, BD Biosciences), leukocytes were washed twice with ice-cold Hanks HEPES buffer, added to resting or activated platelets, and incubated for 20 min at 37°C to generate platelet-leukocyte aggregates. Samples were stained with antibodies against CD45-Percp (BD Bioscience), Ly6G-APC (eBioscience), CD11b-PE (Pharmingen), CD41-FITC (Pharmingen), or isotype control antibodies and then analyzed by flow cytometry (FACSCanto II, BD Biosciences).

The leukocytes infiltrated to the lungs were analyzed by flow cytometry as previously described (Li, Guabiraba, et al., 2014). Briefly, lungs were harvested on day 14 after LLCs administration and digested in 125 mg/ml Liberase TL and 100 mg/ml DNAse 1 (Roche Diagnostics, Switzerland) to characterize the infiltrating leukocytes. Dispersed cells (1 × 10⁶ cells per tube) were stained with fluorochrome‐conjugated mAbs against CD45‐PerCP, CD11b‐APC (all antibodies used in flow cytometry were purchased from BD, NJ, USA. unless otherwise indicated), F4/80‐FITC (eBioscience, CA, USA), and CD206‐PE (BioLegend, CA, USA). Leukocytes were stained with antibodies against ST2–FITC (MD Biosciences, MN, USA), lineage markers (CD3, B220, CD11b, CD11c, FcεR1, SIGLEC‐F) labeled with PE, CD45‐APC, and ICOS‐PerCP (BioLegend) to characterize the infiltrating ILC2s. Cells were analyzed with a BD carlibr Analyzer (BD). Gating strategy (Supporting Information Figure S1) and analysis were performed with FlowJo software (FlowJo LLC OR, USA).

At indicated time points, mice were intraperitoneally anesthetized, tracheotomized, ventilated and intracardially heparinized as described [24] (link). Blood was taken from the caudal Vena cava and lungs were perfused with 0.9% NaCl via the pulmonary artery. Bronchoalveolar lavage was performed twice with 800 µl ice-cold PBS. After spinning, supernatant was snap frozen for cytokine analyses. Total leukocytes were counted manually on Neubauer Chamber and differentiated by fluorescent-activated cell sorter (FACS) analysis (FACS Calibur, BD Biosciences, Heidelberg Germany) using forward versus side scatter characteristics and the specific antibodies CD45 PerCP (clone 30-F11, BD Biosciences), GR-1 PE (clone RB6-8C5, BD Biosciences) and F4-80 APC (clone BM8, Invitrogen, Karlsruhe, Germany) as described [25] (link). Total blood leukocytes were counted and differentiated by FACS analysis using BD TruCOUNT Tubes, forward versus side scatter characteristics and specific antibody staining with CD45 PerCP and GR-1 PE [25] (link). Cytospins from BALF were obtained by centrifugation of 100 µl BALF cell suspension at 20×g for 10 minutes (Cytospin 3, Shandon Ltd, Runcorn, UK) and subsequently stained with May-Grünwald Giemsa.