Pe cy7 anti cd86

The following anti-mouse antibodies were from eBioscience: FITC-anti-F4/80, APC-anti-CD80, PE-Cy7-anti-CD86, PE-anti-MHC-II, PE-anti-CD11b, FITC-anti-Gr-1, APC-Cy7-anti-CD3, PerCP-Cyanine5.5-anti-CD4, APC-anti-CD8a, PE-anti-IFN-γ, and FITC-anti-IL-17. For intracellular cytokine staining, cells were stimulated with PMA (50 ng/ml, Enzo Life Sciences) and ionomycin (1 nM, Enzo Life Sciences) in the presence of brefeldin A (1 mg/ml, Enzo Life Sciences) for 4–5 h. Surface staining was performed in FACS buffer (0.5% BSA, 2 mM EDTA, 0.02% sodium azide in PBS) in the presence of Fc receptor blocking antibody (BioLegend) for 20 min at 4°C. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min at room temperature followed by permeabilization (0.1% BSA, 0.5% saponin in PBS) or with the BD Cytofix/Cytoperm Fixation and Permeabilization Kit. Cytokine staining was performed in permeabilization buffer for 20 min at 4°C. Data acquisition and analysis were performed using BD LSRFortessa X-20 (BD Bioscience) and FlowJo software (Tree Star, Ashland, OR, USA) respectively.

For surface staining, BMDMs or lungs cells were harvested, washed, and stained for 30 min on ice with mixtures of fluorescently conjugated mAbs or isotype-matched controls. mAbs of mice were as follows: FITC-anti-F4/80, APC-anti-CD80, PE-Cy7-anti-CD86, PE-anti-MHC-II, Percp-Cy5.5-anti-CD11b, Pacific Blue-anti-Gr-1 (eBioscience, USA). Cell phenotype was analyzed by flow cytometry on a flow cytometer (BD LSR II) (BD Biosciences, USA). Data were acquired as the fraction of labeled cells within a live-cell gate and analyzed using FlowJo software (Tree Star). All gates were set on the basis of isotype-matched control antibodies.

In vivo, the randomly divided three groups (n = 6) of MI/R induced mice were treated with 200 µL PBS, LM‐miRs or PLM‐miRs, respectively. Two days after administration (the 5th d post injury), phenotypes of heart macrophages were detected by immunofluorescence assay and flow cytometry as well. All experimental procedures are the same as previously described. Alexa Fluor 647‐anti‐CD206 antibody (141 711, BioLegend, USA), Rat‐anti‐F4/80 antibody (ab6640, Abcam, Japan) and Alexa Fluor 488‐anti‐Rat secondary antibody (ab150165, Abcam, Japan) were used for immunofluorescence staining in this section. For flow cytometry, FITC‐anti‐CD45 (#561 867), PerCP‐Cy5.5‐anti‐CD11b (#45‐0112‐82), PE‐anti‐F4/80 (#565 410), PE‐Cy7‐anti‐CD86 (#25‐0862‐82), APC‐anti‐CD206 (#17‐2062‐82) were all purchased from eBioscience (USA). ELISA were adopted to detect the concentration of cytokines (TNF‐α, IL‐1β, TGF‐β, and IL‐10) in cardiac tissue homogenate after PBS, LM‐miRs or PLM‐miRs treatment according to the manufacturer's instruction. Before the detection, the BCA assay was used to quantify the protein concentration of the tissue homogenate, and the subsequent detection results were also corrected according to the corresponding protein concentration.

The proportion of macrophage in the spleen of mice or BMDMs were analyzed by flow cytometry. These cells were stained with APC-CY7-anti-CD45, Fitc-anti-CD11b, PE-anti-F4/80, PE-CY7-anti-CD86, APC-anti-CD206, and their isotype controls (eBioscience, CA) according to the protocol. Flow cytometry was performed with the FACSCalibur (BD Immunocytometry Systems). All data were analyzed using FlowJo software.