Facscalibure flow cytometer

For FACS analysis, all the cells were stained in the presence of an Fc block. For staining, cells were incubated for 20 min at 4 °C using the following antibodies: CD11b-FITC, Ly6C-APC, Ly6G-APC, F4/80- PE, CD11c-PE, CD3-APC, CD8-FITC, CD4-FITC, CD19-FITC (all from eBiosciences, CA, USA) and CD8-PerCP-Cy5.5, CD4-PerCP-Cy5.5 (all from BD Biosciences). The following markers were used to identify specific cell types: monocytes (CD11b+ Ly6C+), neutrophils (CD11b+ Ly6G+), macrophages (F4/80), dendritic cells (CD11c), B cells (CD19), CD4+ T cells (CD3+ CD4+) and CD8+ T cells (CD3+ CD8+).
Unstained cells were used to set up the instrument. Compensation controls were set up using single stains and isotype controls were used to determine the level of non-specific binding. The cells were gated based on simple forward and side scatter patterns. Furthermore, all the dead cells were excluded using the viability dye, propidium iodide and doublet discrimination was performed by plotting FSC-H vs. FSC-A. If we take the example of lymphocytes, they were identified and gated by their forward and side scatter patterns. The CD3+ T cells were then further identified and gated by the expression of CD4+ and CD8+. The expression of surface markers was assessed using CellQuest analysis software on a FACSCalibure flow cytometer (BD Biosciences).

Elisa kits used for measuring cytokines included Human IL-6 Elisa Kit (Diaclone, France) and Human IL-1β Elisa Kit (Zellbio, Germany). Laboratory kits used for measuring the oxidative stress biomarkers (except ROS) included SOD Activity Kit (KSOD96), CAT Activity Kit (KCAT96), Thiol Content Assay Kit (KTHI96), Total Antioxidant Assay Kit (KTAC96), Lipid Peroxidation Kit (KMDA96), and Protein Carbonyl Assay Kit (KPCA96) were purchased from KIAZIST Company, Iran. 2'-7'dichlorofluorescin diacetate (H2DCFDA) (D399, Invitrogen, USA) was used to assess Cellular ROS. Platelet agonist used to activate platelet was thrombin receptor activator peptide (TRAP-6 (SFLLRN), 4,031,274.0005; Bachem, Germany) and antibodies used for flow cytometric measurement of platelet function were FITC Mouse Anti-Human CD62P (Clone AK-4) (555,523, BD Pharmingen, USA) and FITC Mouse IgG1, κ Isotype Control (Clone MOPC-21) (555,748, BD Pharmingen, USA). The instruments used for the laboratory measurements were as follow: 3-30 K Centrifuge (Sigma, Germany), FACSCalibure Flow Cytometer (BD Pharmingen, USA), UV2100 spectrophotometer (UNICO, China), Infinite M200 plate reader (Tecan, Austria), and ELISA reader apparatus (BD Biosciences, USA).