Apc anti human cd11b

The tMACs or iMACs were stimulated with LPS and IFN-γ for the indicated time. The single-cell suspensions were then prepared and incubated with an antibody or antibody cocktails for 15 min at room temperature for cell surface staining. Antibodies used in this study were PE Mouse IgG1, κ isotype (Biolegend, Cat: 400113, Clone: MOPC-21, Lot: B245984), APC Mouse IgG1, κ isotype (Biolegend, Cat: 400119, Clone: MOPC-21, Lot: B243042), FITC Mouse IgG1, κ isotype (Biolegend, Cat: 400107, Clone: MOPC-21, Lot: B199152), APC anti-human CD206 (Biolegend, Cat: 321109, Clone: 15-2, Lot: B348965), APC anti-human CD86 (Biolegend, Cat: 305411, Clone: IT2.2, Lot: B351349), PE anti-human CD80 (Biolegend, Cat:305208, Clone: 2D10, Lot: B330518), PE anti-human CD163 (Biolegend, Cat: 333606, Clone: GHI/61, Lot: B347256), FITC anti-human CD14 (Biolegend, Cat: 325604, Clone: HCD14, Lot: B268830) and APC anti-humanCD11B (Biolegend, Cat: 301309, Clone: ICRF44, Lot: B278346). These antibodies were used at a 1:100 dilution. Data were recorded on Beckman DxFLEX and analyzed with the FlowJo V10 software.

After polarization of THP1 cells into M2-like macrophages, cells were digested using Accutase (BioLegend, 423201). After centrifugation, cells were resuspended using 100 μl PBS, and incubated for 5-10 min at 4 °C using FcRblock (1 μg/test, 101319, BioLegend). Subsequently, staining was performed using APC anti-human CD11b (0.5 μg/test, 101212, BioLegend), PE/Dazzle™ 594 anti-human CD86 (5 μl/test, 305433, BioLegend), and PE anti-human CD206 (5 μl/test, 321105, BioLegend) and incubated at 4 °C for 60 min. Wash the cells 2 ~ 3 times, add 500 μL cell staining buffer to resuspend the cells, and detected with FACSAria™ III (BD Biosciences CA, USA).

The polarization-related surface markers of RAW264.7, MH-S, and THP-1 macrophages were detected by flow cytometry. In our previous study (5 (link)), we found that 10 μg/ml CSE-EVs could significantly promote M1 polarization of RAW264.7 macrophages, thus we treated macrophages (5×10⁴ cells/cm²) with 10 μg/mL EVs in EVs-free RPMI 1640 mediums for 24 h in this study. For positive control, 2 μM SF1670 and 1 μM AG14361 were used to inhibit PTEN and PAPR1 expression of THP-1 cells, respectively. Then, the cells were collected and stained with the following antibodies on ice for 20 min in the dark. APC anti-mouse CD86, APC anti-mouse CD80, APC anti-human CD11b, PE anti-human CD86, and PE anti-human CD80 (Biolegend, San Diego, CA, USA) were used in this procedure. After being washed twice with PBS, cells were fixed with fixation buffer and then analyzed by flow cytometry (CytoFLEX, Beckman).

Hypoxia green reagent; pHrodo Green E. coli; DAF-FM DA; JSH-23; QNZ; ABT-263; and ABT-737 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The fetal bovine serum was from Gibco (Gibco, Sigma Aldrich Company Ltd., Waltham, MA, USA). Intracellular staining permeabilization wash buffer; Human TruStain FcX (Fc Receptor Blocking Solution); PE anti-human Ki-67; isotype control antibodies PE Mouse IgG1 k isotype Ctrl; APC Mouse IgG1 k isotype Ctrl; FITC Mouse IgG1 k isotype Ctrl; PE Mouse IgG2a k isotype; PE Mouse IgG2b k isotype and antibodies APC anti-human CD11b; PE anti-human CD284; PE anti-human CD36; PE anti-human CD33; PE anti-human HLA-DR; PE anti-human HIF-1α were from BioLegend (San Diego, CA, USA). MycoFluor™ Mycoplasma Detection Kit was from Molecular Probes Inc. (Eugene, Oregon, USA). FITC anti-human CD68 was from BD Bioscience (Franklin Lakes, NJ, USA). Culture medium RPMI 1640; 2-mercaptoethanol; PBS; Calcein AM; Bisbenzimide Hoechst 33342 (H33342); propidium iodide (PI), resazurin; LPS of E. coli O111: B4; antibodies FITC anti-human CD11c; FITC anti-human CD14; FITC anti-human CD54; NF-kB activation inhibitor IV, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

APC/Cy7 anti-human CD15 (Biolegend, UK) was used to discriminate neutrophils in whole blood staining. FITC anti-human CD62L, PE anti-human CD18, and APC anti-human CD11b (Biolegend, UK) were used to detect surface expression of adhesion molecules. Irrelevant antibodies of the same isotypes were used as negative controls.