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5 protocols using percp conjugated cd45

1

Cardiac Leukocyte Characterization by Flow Cytometry

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Total cardiac cells were gated on PerCP-conjugated CD45 (BD Biosciences), and the following antibodies were used: PE-Cy7–conjugated anti-CD11b (BD Pharmingen), PE-conjugated anti-Ly6G (lymphocyte antigen 6 complex, locus G; 1A8; BD Pharmingen), FITC-conjugated anti-Ly6C (lymphocyte antigen 6 complex, locus C; Biolegend), PE-Alexa700–conjugated anti-CD3 (eBioscience) and APC-conjugated anti-F4/80 (Bio-Rad). CD45+/CD11b+ leukocytes were gated on F4/80 monocytes and then further stratified by Ly6G and Ly6C expression. Monocytes were identified as CD11bhighLy6GLy6C+. Neutrophils were identified as CD11b+Ly6Ghigh and T cells as CD45+CD3+. The total number of cells was then normalized to LV weight. Cells were analyzed using a flow cytometer (LSR II; BD Biosciences).10 (link),11 (link)
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2

Immunophenotyping of Trophoblast Cells

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TNCs (2 × 105) were labeled with a combination of PerCP‐conjugated CD45 (BD Biosciences, clone 2D1) and APC‐conjugated CD34 (BD Biosciences, clone 8G12) plus: FITC‐conjugated CD14 (BD Biosciences, clone MSE2) for monocyte detection; PhycoErythrin (PE)‐conjugated CD56 (BD Biosciences, clone MY31), FITC‐conjugated CD2 and CD3 (BD Biosciences, clone MOPC21 and UCHT1), FITC‐conjugated CD19 and CD20 (BD Biosciences, clone HIB19 and 2H7) for lymphocyte detection; and FITC‐conjugated CD15 (BD Biosciences, clone MMA) for granulocyte detection. The percentage of cell impurities was calculated as the percentage of CD45+ events after the exclusion of CD34+ events using a FACS Canto II analyzer (BD Biosciences) and FACS DIVA software (Supporting Information Fig. S2).
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Lung Leukocyte Phenotyping in Mice

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The phenotype of cells from mouse lungs was determined by incubating lung leukocytes with the indicated antibodies and CD16/32 to limit nonspecific binding. Leukocytes were stained at 4°C for 15 min in PBS containing 1%BSA and 0.01% sodium azide. Cells were stained with combinations of the following antibodies: FITC-conjugated I-Ab; PE-conjugated CD11c; PerCP-conjugated CD45; and APC-conjugated F4/80 and EpCAM from BD Biosciences. For intracellular IL-33 staining, cells were incubated with Cytofix/Cytoperm (BD Biosciences, San Diego, CA), washed in Permeabilization Buffer (BD Biosciences), and stained for 30 min with PE-conjugated IL-33 (R&D systems, Minneapolis, MN). Cells were washed and resuspended in 1% paraformaldehyde. Isotype controls were used. Data was acquired using BD Accuri™ C6 cytometer and analyzed using FCS Express 4.0 Software. For cell sorting experiments, F4/80+ leukocytes from the lungs of WT and CCR2−/− mice were isolated at day-7 p.i. using 5-laser FACS Aria II (BD Biosciences).
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4

Pluripotency and Hematopoietic Marker Analysis

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For analysis of pluripotency markers, hiPSCs were dissociated into single cells using TrypLETM Select (Gibco), blocked with 10% human AB serum, stained with FITC-conjugated SSEA-3, PE-conjugated SSEA-4, and Alexa Fluor 647- conjugated TRA-1-60 (BioLegend, San Diego, CA, United States), and analyzed by BD FACS Canto (BD Biosciences, Franklin Lakes, NJ, United States).
For immunophenotypic analysis of the differentiated HSPCs, cells were resuspended in FACS buffer (PBS with 2% BSA), incubated with antibody cocktail for 30 min at 4°C in the dark, and analyzed by BD FACS Canto. The antibodies used in this study included FITC-conjugated CD34, PerCP-conjugated CD45, PE-Cy7-conjugated CD43 (BD Biosciences), and APC-conjugated CD235a (glycophorin A, GPA) (BioLegend). Absolute counts were performed using BD Trucount tubes containing a known quantity of fluorescent beads.
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5

Lung Leukocyte Phenotyping in Mice

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The phenotype of cells from mouse lungs was determined by incubating lung leukocytes with the indicated antibodies and CD16/32 to limit nonspecific binding. Leukocytes were stained at 4°C for 15 min in PBS containing 1%BSA and 0.01% sodium azide. Cells were stained with combinations of the following antibodies: FITC-conjugated I-Ab; PE-conjugated CD11c; PerCP-conjugated CD45; and APC-conjugated F4/80 and EpCAM from BD Biosciences. For intracellular IL-33 staining, cells were incubated with Cytofix/Cytoperm (BD Biosciences, San Diego, CA), washed in Permeabilization Buffer (BD Biosciences), and stained for 30 min with PE-conjugated IL-33 (R&D systems, Minneapolis, MN). Cells were washed and resuspended in 1% paraformaldehyde. Isotype controls were used. Data was acquired using BD Accuri™ C6 cytometer and analyzed using FCS Express 4.0 Software. For cell sorting experiments, F4/80+ leukocytes from the lungs of WT and CCR2−/− mice were isolated at day-7 p.i. using 5-laser FACS Aria II (BD Biosciences).
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