Macsquant q10 cytometer

Synovial fluid mediators were determined by screening pro-inflammatory cytokines on three SF using a membrane-based Human Cytokine Array kit (ARY005B, R&D Systems, Minneapolis, MN, USA). The chemiluminescent signals were visualized with a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA) and analyzed using Image Lab 5.0. IL-6 and IFN-γ levels were quantified by ELISA according to the manufacturer's instructions (R&D Systems). Concentrations of MCP-1 levels were determined by Cytometric Bead Array (BD Biosciences, San Diego, CA, USA). Data was acquired on an LSRII cytometer (BD Biosciences) and analyzed using FCAP array v3 software (BD Biosciences). Quantification of IL-1β, IL-23, IL-12p70, IL-12p40, CCL17, CXCL10, IL-10, and IL-1RA in SF was performed by the LegendPlex Human M1/M2 Macrophage Panel (BioLegend, San Diego, CA, USA). Data was acquired on a MACSQUANT Q10 cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) and analyzed using Legendplex Data Analysis Software (BioLegend).
IL-6 and TIMP3 were quantified in ADSC culture supernatants by ELISA (R&D Systems).

Blood samples were collected from all rats in each group by clean arteriopuncture into 2 ml vacutainer tubes under strict sterile conditions. The samples were centrifuged a 1000 rpm for 20 min to obtain platelet-rich plasma (PRP). Then, the PRP was centrifuged at 12000 rpm for 5 min to prepare platelet-poor plasma (PPP). Phenylethylamine (PE) anti-Rat CD31-PECy7 (eBioscience, U.S.A.) and allophycocyanin (APC) labeled anti-mouse/rat CD42b (Biolegend, Germany) were used to detect endothelial microparticles (EMPs). About 50 μl PPP were incubated with 8 ml of each labeled antibodies for 30 min at 4°C in the dark. The stained samples were resuspended in 0.2 ml of PBS and then kept for flow cytometry analysis.
For flow cytometric detection of vascular cell adhesion molecule (VCAM)-1, intracellular adhesion molecule (ICAM)-1, and selectin P (CD62P) in cavernous tissue. The cavernous tissue was digested to prepare 1 × 10⁶/ml single-cell suspension. Cells were incubated for 30 min in PBS containing anti-rat CD106-PE (VCAM-1), anti-rat CD54-PE (ICAM-1), and anti-mouse/rat CD62P-APC (P-selectin). The stained cells were then detected by the MACSQUANT Q10 cytometer (Miltenyi Biotec) and analyzed using FlowJo v7.6.5 software (Tree Star).

For intracellular cytokine detection, T cells were restimulated with PMA (50 ng/mL) and ionomycin (1 μg/mL, Sigma-Aldrich) in the presence of Brefeldin A (0.2%, BD Biosciences) for 4 h. Cells were then stained with antibodies against T cell markers or their respective isotype control anti-CD25-PECy7 and anti-CD4-FITC (BD Biosciences) for 20 min at 4°C in the dark, fixed, and permeabilized with the Transcription factor buffer set (BD Biosciences) and then stained intracellularly with anti-Foxp3-PE, anti-IFNγ-PE, and anti-IL17A-AF647 antibodies (BD Biosciences) for 30 min at 4°C for flow cytometry detection of Tregs, Th1, and Th17 cells, respectively.
For flow cytometric detection of macrophages, cells were stained with the following antibodies or their respective isotype controls anti-CD40-APCy7 (BioLegend), anti-CD80-BV421 (BioLegend), anti-CD16-V500 (BD Biosciences), anti-CD206-AF488 (BioLegend), anti-CD163-APC (Miltenyi Biotec) for 20 min at 4°C in the dark. All samples were acquired on a MACSQUANT Q10 cytometer (Miltenyi Biotec) and analyzed using FlowJo v7.6.5 software (Tree Star).