Flowcytomix mouse th1 th2 kit

A flow cytometry-based technique was used for cytokine quantitation (IFN-γ, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10 and IL-17) from mice sera. A FlowCytomix Mouse Th1/Th2 kit (Bender MedSystems GmbH, Vienna, Austria) was used according to the manufacturer’s instructions. Briefly, different sized fluorescent beads, coated with capture antibodies specific for the aforementioned cytokines were incubated with mouse sera samples and with biotin-conjugated secondary antibodies for 2 h at room temperature. The specific antibodies bind to the analytes captured by the first antibodies. After washing the tubes with PBS plus 2% fetal calf serum, Streptavidin-Phycoerythrine (S-PE) solution was added and incubated at room temperature for 1 h. S-PE binds to the biotin conjugate and emits fluorescent signals. Flow cytometry data were collected using a FACSCalibur flow cytometer (BD Biosciences) (8000 events were collected, gated by forward and side scatter), and data were analyzed using FlowCytomix Pro 3.0 software (Bender MedSystems, Vienna, Austria). Each cytokine concentration was determined from standard curves using known mouse recombinant cytokine concentrations.

Serum samples were analyzed for the presence of cytokines (IL-1alpha, IL-2, IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha, granulocyte monocyte colony-stimulating factor (GM-CSF), IL-4 and IL-17) using a FlowCytomix mouse Th1/Th2 kit (Bender MedSystems) according to the manufacturer’s instructions. Briefly, fluorescent beads coated with antibodies against to the specified cytokines were added to serum samples and incubated with biotinylated secondary antibodies. The amount of cytokine that bound to the antibodies was recognized by streptavidin-phycoerythrin conjugate and then detected using a flow cytometer (FACScan, Becton Dickinson, San Diego, CA, USA). Cytokine levels were determined by comparison with a standard curve (concentration range for all cytokines 0-20000 pg/mL) using FlowCytomix Pro 2.4 software.