Cd163 apc

PBMCs were incubated with mixtures of fluorochrome-conjugated antibodies, and identified by flow cytometry. Data acquisition and analysis were performed using a BD CANTO II Flow Cytometer and BD FACS DIVA software (BD Biosciences, Flanklin Lakes, New Jersey). Dead cells were distinguished by 7-amino-actinomycin D (7-AAD, BD Biosciences) staining. Cells were not permeabilized for intra-staining. To identify monocyte lineage cells, the surface markers CD14-PE (Biolegend, San Diego, California, clone HCD14) and CD16-APC/Cy7 (Biolegend, clone 3G8) were used. Pro-inflammatory and anti-inflammatory phenotypes were characterized using S100A9-FITC (BioRad, Hercules, California, clone MAC387) and CD163-APC (Biolegend, clone RM3/1) antibodies (Supplementary Table). S100A9 and CD163 expression levels were determined using a positive dataset for each antibody, identified using a matched concentration of mouse IgG1 kappa isotype control for fluorochrome color (Biolegend, clone MOPC21).

We used heparin sodium anticoagulation tubes to collect 5 ml of peripheral blood, and then took several test tubes. Add antibody PI (10 μl)/CD45-cyanine7 (1 μl)/IgG2b-CY5.5 (1 μl)/IgG1-APC (1 μl) (Biolegend Corp., San Diego, CA, USA) to each tube of 100 μl whole blood as a negative control group. PI(10 μl)/CD45-cyanine7(1 μl)/CD86-CY5.5(1 μl)/CD163-APC(1 μl) (Biolegend Corp., San Diego, CA, USA) was used as the experimental group. Add 1 ml of hemolysin to each test tube. The target cell gate can be set more accurately in the FS/SS graph of the flow cytometer. In CD45/SS flow cytometry, the monocyte gate can be set more precisely. Using a three-color scheme, select the CD45/SS map to set a gate, and detect high expression of CD14 (macrophages) in the blood. As per the antibody manufacturer's instructions, human M1 and M2 macrophages were expressed by adding fluorescently labeled mouse antihuman CD14-FITC, CD86-PE-Cyanine5, and CD163-APC monoclonal antibody reagents (10 μL; Biolegend Corp., San Diego, CA, USA). The mixtures were mixed well and allowed to react in a refrigerator at 4°C for 30 min. The mixtures were centrifuged at 1500 rpm for 5 min to remove the supernatants. The obtained cell deposits were resuspended in PBS for flow cytometry analysis.

The expression levels of M1 and M2 macrophage polarization markers CD86, CD163, and CD206 were detected by flow cytometry. In 100 μL of peripheral blood anticoagulated with heparin sodium, we added mouse anti-human CD14-FITC, CD86-PE-Cyanine5, CD163-APC, and CD206-PE (Biolegend, San Diego, CA, USA) to 10 μL respectively. After the sample was mixed well, it was incubated in the dark at room temperature for 20 min, followed by the addition of red blood cell lysate. Further, each sample was shaken and incubated in the dark at room temperature for 15 min, followed by flow cytometry measurements. Then, the forward scatter (FSC) was taken as the abscissa and the longitudinal scatter as the ordinate (SSC) to develop a scatter plot. The lymphocyte gate was set, and then the boundary fluorescence marker was determined. The results were analyzed with Flow Jo 7.6.1 software.