Cytometric bead arrays kit

Peripheral blood samples were collected before and immediately after exercise for each volunteer, and plasma was prepared and stored at −80°C until required. IL-8 level was measured using the cytometric bead arrays kit (BD Bioscience, USA) according to the manufacturer's protocol. Samples were acquired in a FACSCanto flow cytometer (BD Biosciences) and analyzed using the FCAP Array v1.0.1 software (Soft Flow Inc., Hungary). The detection limits were 0.2 pg/mL for IL-8 level.
Adiponectin, plasma soluble TNF receptor (sTNFR1, sTNFR2), leptin, resistin, and BDNF levels were measured using conventional sandwich enzyme-linked immunosorbent assay kits (DuoSet, R&D Systems, USA), according to the manufacturer's instructions. The detection limits were 5.0 pg/mL for all kits.

Twenty-four hours after the initial assessment, six milliliters of each of the blood samples was collected in a local laboratory following 3 hours of fasting in the morning. The tubes (sodium heparin and serum) were centrifuged to remove cells and debris and were stored as plasma, serum and erythrocytes aliquots at -80°C until use.
Plasma leptin, adiponectin, resistin, BDNF and soluble TNF receptor (sTNFR1, sTNFR2) levels were measured using conventional sandwich ELISA kits (DuoSet, R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. The detection limits were 5.0 pg/mL for all the kits.
Plasma chemokines (CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL10/IP-10 and CXCL9/MIG) were measured using the cytometric bead arrays kit (BD Bioscience, San Jose, CA, USA) according to the manufacturer’s protocol. Bead flow cytometry allows the simultaneous quantification of various proteins in the same test. Samples were acquired in a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using the FCAP Array v1.0.1 software (Soft Flow Inc.). The detection limits were 0.2 pg/mL for IL-8, 1.0 pg/mL for RANTES, 0.2 pg/mL for MIG, 2.7 pg/mL for MCP-1 and 2.8 pg/mL for IP-10.
Serum cortisol was measured using competitive ELISA kit (IBL, AMERICA, Minneapolis, MN, USA) according to manufacturer’s instructions. The detection limits were 55.2 nmol/L.

Blood was drawn at 8 a.m. (10 mL from the antecubital fossa of the upper limb with disposable material) after participants fasted from food and drink and without using medication for 10 h. The samples were drawn in vacutainer bottles with heparin in a sterile environment. Immediately after this procedure, the samples were centrifuged at 3000 rpm in a centrifuge for 10 min. Plasma samples were extracted and kept at − 80 °C for 6 months before being analyzed. The IL-2, IL-4, IL-5, IL-10, TNF, adiponectin, leptin, resistin, BDNF, sTNFr-1 and sTNFr-2 levels were analyzed by Enzyme-linked Immunosorbent technique (ELISA) (Duo-Set, R&D Systems, Minneapolis, USA). The plasma levels of IL-6, IL-8, and IFN were measured using the cytometric bead arrays kit (BD Bioscience, San Jose, CA) according to the manufacturer’s protocol. Samples were acquired in a FACSCanto flow cytometer (BD Bioscience) and analyzed using the FCAP array v1.0.1 software (Soft Flow)^{20 (link)}.