Percp cy5.5 anti cd11b

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.

For flow cytometric analysis of tumour tissues, single cell suspensions were first prepared. The tumour masses were harvested from mice, cut into small pieces, and digested in DMEM medium containing collagenase A (1 mg/ml) (#10103586001, Roche) and collagenase D (1 mg/ml) (#11088866001, Roche) at 37°C for 30 min, and were then filtered with 70‐mm cell strainers (Becton and Dickinson).
The cells were stained with fluorescence‐labelled antibodies: PE‐anti‐CD3e (#553064), BV421‐anti‐CD45.2 (#560697), BUV395‐anti‐NK.1.1 (#564144), APC‐Cy7‐anti‐CD11c (#561241), AmCyan‐anti‐MHC‐II (#562366), BUV661‐anti‐CD4 (#612974), BV786‐anti‐CD8 (#563332). (all from BD Biosciences); FITC‐anti‐Ly 6C (#MCA2389A488) and APC‐anti‐CD206 (#MCA2235A647T) from (Bio‐Rad AbD Serotec); anti‐F4/80 (#123113), PE‐Texas Red‐anti‐Ly‐6G (#127647) were from Biolegend, PerCP‐Cy5.5‐anti‐CD11b (#45‐0112‐80) was from eBiosciences, , FVs510 Viability Staining Solution was used to exclude dead cells. Acquisition was performed on the BD FACSFortessa (BD Biosciences) and analysed using FlowJo (BD Biosciences) software.