Mouse cytokine array panel a kit

Cells were stimulated with SH005S7 at the indicated concentrations for 48 hours. The cell culture medium was collected and then stored at −80°C. The supernatants were tested for cytokine expression profiles, using the mouse cytokine array panel A kit (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer's instructions.

The cytokine/chemokine profiles of BMCs supernatants were assessed by ELISA-based cytokine array by using Mouse Cytokine Array Panel A kit (R&D Systems, Milano, Italy) accordingly to the manufacturer's instructions. Immunoreactive dots were visualized using LiteAblot Turbo luminol reagents (Euroclone, Milano, Italy) and Hyperfilm-ECL film (Euroclone, Milano, Italy) and quantitated densitometrically.

Profiling of cytokines and chemokines was conducted using a multiplex cytokine array. Briefly, protein extracted from the substantia nigra of WT and DJ-1 KO mice was quantified by using a BCA protein assay kit (Pierce, Rockford, IL), followed by analysis with a mouse Cytokine Array Panel A kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instruction. Proteins (200 μg / array) were loaded onto the surface of the arrays for binding the primary antibodies coated on the array surface, washed to remove the unbound proteins, and incubated with HRP-conjugated secondary antibodies to form sandwich complexes. After that, the array membranes were washed again and the arrays were developed using an enhanced chemiluminescence (ECL) substrate. The array images were then captured by a charge-coupled device (CCD) imaging and documentation system (UVP, Upland,. CA, USA). Levels of protein spots in both WT and KO groups were measured by using ImageQuant 5.0 software, and normalized against the corresponding protein spots in the WT group.

Cell culture media were harvested 24h after LPS treatment from the bottom wells. One ml of each cell culture medium supernatant was collected by centrifugation at 500 x g for 5 minutes to remove particulates. Cytokines in the culture supernatant were analyzed by Mouse Cytokine Array Panel A kit (Cat# ARY006, R&D Systems, Inc. Minneapolis, MN) by following the instructions provided by the manufacturer. This array contains 40 different capture antibodies printed in duplicate to simultaneously detect differences in relative levels of cytokines and chemokines between samples. Positive signals detected by chemiluminescent reagents in the kit were exposed to the X-ray film. The pixel densities of the signals were determined by densitometric scanning using an HP Scanjet G3010 Photo Scanner and Image J V1.40 (NIH, MD). The average pixel density of the pair of duplicate spots representing each cytokine expression levels was determined by subtracting an average background signal (negative control spots as a background value) from each spot.

Secreted cytokines were measured using a mouse cytokine array panel A kit (R&D systems) and following the manufacturer’s protocol. Conditioned media of pancreatic cancer cell lines were collected as supernatants. The detection antibody cocktail was added to the supernatants for 1 h followed by incubation with membranes overnight at 4 °C. After washing, HRP-conjugated Streptavidin was added, and membranes were exposed with Chemi Reagent. Images were taken with an Amersham™ Imager 600 (GE Healthcare).