Pe anti human cd86

Peripheral blood mononuclear cells were stimulated with either a 1, 10, or 25 μg/mL suspension of OMVs from both strains or a 5 μg phytohemagglutinin (PHA) solution for 12, 24, and 48 h. Plates were incubated at 37°C under 5% CO₂. Monoclonal antibodies (mAbs) coupled to the following fluorochromes were used: anti-human CD91-eFluor 660 (eBioscience), anti-human CD3-APC (BD Pharmingen^TM), anti-human CD19-PE-Cy7 (BD Biosciences), anti-human PD-L1-PE (BioLegend), anti-human PD-1-FITC (BioLegend), anti-human CD86-PE (BioLegend), anti-human CD69-TRI-COLOR (Molecular Probes). After stimulation, 100 μL of supernatant were collected from wells and stored at −70°C until usage. Then, PBMCs were collected and washed with FACS buffer, followed by incubation with diluted mAbs in FACS buffer for 1 h at 4°C (protecting the reaction from light). Finally, cells were washed with FACS buffer, fixed with PBS-PFA (Paraformaldehyde) 1%, and resuspended in 400 μL of FACS buffer. Samples were analyzed in LSRFortessa^TM cytometer (BD Biosciences), providing a total number of 30,000 events, and the resulting data was analyzed with the aid of the FlowJo^® software.

The expression of CD73, CD90, CD105, CD34, CD45, and CD11b in GMSCs and CD11b, CD206, and CD86 in PBMC-derived macrophages were analyzed using the FACS Calibur (Becton Dickinson, USA) and the CellQuest software (Becton Dickinson). The adherent cells were washed with PBS and collected using Accutase (Nacalai Tesque) and resuspended in 50 μL staining buffer (BD Pharmingen, country). The harvested cells were blocked with 2 μL Human Trustain FcX (Fc receptor Blocking Solution; BioLegend) for 10 min at room temperature, and stained with 2 μL antibodies namely, FITC anti-human CD73, FITC anti-human CD90, Alexa Fluor® 488 anti-human CD105, FITC anti-human CD34, PE anti-human CD45, FITC anti-human CD11b, Alexa Fluor® 488 anti-human CD11b, PE anti-human CD206, APC anti-human CD206, PE anti-human CD86 and APC anti-human CD86 (BioLegend) in the dark for 30 min at 4 °C.

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).

For flow cytometry analysis, THP-1 cells, RAW264.7 cells, and mouse bone marrow macrophages were seeded in a 6-well plate at a density of 1 × 10⁶ cells/well. After stimulation with IFNγ, or IFNγ after rM180 treatment for 24 h, cells were detached with Accutase (Nacalai Tesque), centrifuged at 300 g for 5 min, and washed with stain buffer (BD Pharmingen, San Diego, CA, USA). Cells were blocked with human TrusStain FcX (BioLegend) for 10 min at room temperature. To determine the surface expression of target molecules, cells were then washed and stained with PE anti-human HLA-DR (BioLegend), PE anti-human CD86 (BioLegend), Alexa Fluor 488 anti-human HLA-A, B, C (BioLegend), and FITC anti-mouse I-A^d (BioLegend). Isotype controls were used to confirm antibody specificity. Cells were incubated in the dark for 30 min at 4°C and analyzed using a BD FACSVerse flow cytometer (BD Biosciences, San Diego, CA, USA). Data were processed using FlowJo^TM (v10.5.3) software (BD Biosciences).

Flow cytometry was used to detect the surface markers of THP-1 cells that had been stimulated for 24 hours and collected from 6-well plates. The suspension, which had 1 × 10⁶ M1-polarized and M2-polarized cells, was then divided into 1.5 ml EP tubes and incubated with the following antibodies (PE anti-human CD86 and APC anti-human CD206; both from BioLegend, San Diego, CA, USA) on ice for 30 min in the dark. Flow cytometry (BD Biosciences, San Diego, CA, USA) was used to detect cells that had been suspended in 500 μl PBS with 3% FBS after being washed twice.

THP-1, the most widely used model for the human monocytes/macrophages, was obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China), and routinely cultured in RPMI 1640 medium (HyClone, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. We polarized THP-1 monocytes according to the procedure reported previously [25 (link)]. THP-1 were firstly differentiated into M0 macrophages with 100 ng/ml phorbol 12-myristate 13-acetate (PAM, Sigma, USA) for 24 h. M0 cells were then polarized into M1 macrophages with incubation with 100 ng/ml LPS (PeproTech, USA) and 20 ng/ml IFN-γ (PeproTech) for an additional 48 h. To generate M2 phenotype, M0 populations were exposed to 20 ng/ml IL-4 (PeproTech) and 20 ng/ml IL-13 (PeproTech) for another 48 h. The phenotypes of polarized macrophages were determined by cell makers via flow cytometry. M1 and M2 macrophages were stained using lineage-specific antibodies, with PE anti-human CD86 for M1, APC anti-human CD206 (BioLegend) for M2, and FITC anti-human CD11b (BioLegend) for all monocytes. FlowJo software was used for statistical analysis.