Flowcytomix simplex kit

Concentrations of sOPN, sCD44-v6 and sVCAM-1 in samples were measured separately using a FlowCytomix Simplex Kit (eBioscience, Vienna, Austria). The kit consists of fluorescent microspheres with an emission wavelength at 700 nm (5 μm diameter for sOPN and sCD44-v6 analysis and 4 μm diameter for sVCAM-1 analysis). Microspheres are coated with specific antibodies raised against each of the analytes (sOPN, sCD44-v6 or sVCAM-1). They also contain a biotin-conjugated second antibody and streptavidin-phycoerythrin emitting at 575 nm. Samples were run on a Cell Lab Quanta™ SC-MPL (Beckman Coulter, Fullerton, United States). Electronic volume versus side scatter gating was employed to exclude any sample particles other than 5 μm (4 μm) microspheres. Samples were acquired by Cell Lab Quanta^TM SC-MPL software (Beckman Coulter, Fullerton, United States) and analyzed using Flowcytomix™ Pro 3.0 software (eBioscience, Vienna, Austria). The lower limits of detection of sOPN, sCD44-v6 and sVCAM-1 were 0.432 ng/ml, 0.126 ng/ml and 0.9 ng/ml, respectively.

Concentrations of sVCAM-1 in samples were measured using a FlowCytomix Simplex Kit (eBio-science, Vienna). The kit consists of fluorescent microspheres (diameter: 4 μm, emission wavelength at 700 nm) coated with specific antibodies raised against sVCAM-1. It also contains a biotin-conjugated second antibody and straptavidine-phycoerythrin emitting at 575 nm. Samples were run on a Cell Lab Quanta^TM SC-MPL (Beckman Coulter). Samples were acquired by the Cell Lab Quanta^TM SC-MPL software (Beckman Coulter) and analysed using Flowcytomix^TM Pro 3.0 software (eBioscience). Electronic volume vs. side scatter gating was employed to exclude any sample particles other than 4 μm microspheres. A seven point standard curve ranging from 2.74 to 2000 ng/ml was obtained by serial dilution of the reconstituted lyophilized standard. The lower limit of detection was 0.9 ng/ml.

Concentrations (pg/mL) of IL-1α, IL-1β, IL-6, and TNF-α in serum were measured by using a commercial multiplex suspension array technology kit (Mouse IL-1α, IL-1β, IL-6, and TNF-α FlowCytomix simplex kit, eBioscience) and flow cytometry. 25 μL of serum was tested following manufacturer's instructions. MFI from microspheres was acquired with a BD FACSCanto II and analyzed in FlowCytomix Pro2.2.1 software (eBioscience). Concentration of each analyte was obtained by interpolating fluorescence intensity to a 7-point dilution standard curve supplied by the manufacturer. Any value below the limits of detection was given zero for that cytokine.

Before operation four ml of peripheral blood were collected in a vacutianer, without anticoagulant or other additives and used for sVCAM-1 analysis. Twenty ml of ascites were aspirated into a sterile syringe at the beginning of each operation and immediately transferred into a conical tube and kept on ice until centrifugation at 1000 x g for 10 min at 4 °C. Serum was separated by centrifugation at 2000 x g for 15 min at 4 °C. Ascites supernatants and serum were stored in aliquots at − 80 °C. sVCAM-1 sample concentration was analyzed by means of flow cytometric bead-based assay and measured using a FlowCytomix Simplex Kit (eBio-science, Vienna). The kit consists of fluorescent microspheres (diameter: 4 μm, emission wavelength at 700 nm) coated with specific sVCAM-1antibodies raised.