Anti il 10 apc

Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Cryopreserved PBMCs from each participant at all time points were investigated simultaneously. After overnight rest of thawed PBMC, the cells were cultured in the presence of anti-CD107a-PE in complete RPMI containing 2 μg/mL of recombinant HBsAg (MyBioSource, USA) and 5 μg/mL of PHA (Sigma-Aldrich, USA) as the positive stimulation control or complete media alone as the unstimulated control at 37°C in a 5% CO₂ incubator for 16–18 hr.
Cytokine-producing T cells analysis was performed as described previously [34 (link)]. Briefly, the overnight culture was further incubated with 5 μg/mL of brefeldin A and 1 μM of monensin (Sigma-Aldrich, USA) for 4 hr. Then, the cells were stained with anti-CD8-PE Alexa Fluor 610 (Life Technologies, USA), and anti-CD4-APC/Cy7 and anti-CD45RO-Pacific Blue (BioLegend, USA). After fixation and permeabilization, intracellular staining was performed by incubating the cells with anti-CD3-Krome Orange (Beckman Coulter, USA), and with anti-TNF-α-FITC, anti-IFN-γ-PerCP/Cy5.5, anti-IL-2-PE/Cy7, and anti-IL-10-APC (BioLegend, USA). At least 100,000 lymphocytes were collected for each sample by using Cyan ADP 9-color flow cytometer (Beckman Coulter, USA). Flow cytometric analysis of cytokine-producing or degranulation maker CD107a-expressing T cells was performed by using Kaluza software (Beckman Coulter, USA).

To analyze the immunomodulatory potential of the treatments on Th1, Th2, and Th17 cytokine production, the Tconv cells were cultivated in the presence of MSCs or their exosomes, followed by intracellular staining. In summary, differentiated cells were washed once with PBS and resuspended in 100 μl of staining buffer (PBS + 2% FBS). The cells were then fixed and permeabilized by LEUCOPERMTM (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s manual. The antibodies including anti-IFN-γ-PE-Cy7, anti-IL-17A-PE (eBioscience, San Diego, CA, USA), and anti-IL-10-APC (BioLegend, San Diego, CA, USA) were used for staining and the cells were evaluated by flow cytometry.

Femoral bone marrow cells were isolated and plated, consistent with methods reported for In Vitro Assays. Whole marrow cultures were maintained undisturbed for 4-hours; cultures were subsequently stimulated for 1-hour with 1 µl/ml PMA and 2 µl/ml Ionomycin, followed by 4-hours stimulation with 1 µl/ml Monensin. Cells were harvested, washed, and counted. Surface stains were performed and intracellular stains were carried out following the fixation-permeabilization buffer manufacturer’s protocol (BioLegend): anti-CD3e-FITC (Tonbo Biosciences, clone 145-2C11), anti-CD4-APC/Cy7 (BD Pharmingen, clone GK1.5), anti-CD8a-PerCP/Cy5.5 (BioLegend, clone 53-6.7), anti-IL10-APC (BioLegend, clone JES5-16E3), anti-IL17a-PE (eBioscience, clone eBio17B7), anti-IFNγ-BV421 (BioLegend, clone XMG1.2). Data was acquired by the FACSVerse System (BD Biosciences). Analyses were performed via FlowJo VX software.