Proteome profiler mouse cytokine array panel a

For the analysis of the secreted cytokines in the supernatant of wildtype and Vav3^–/– astrocytes, the Proteome Profiler Mouse Cytokine Array Panel A (R&D Systems, Cat. No.: ARY006) was used without changing the manufacturers protocol. Here, the astrocytes were cultured in T-25 flasks (Sarstedt; Cat. No.: 83.3911.002) which were previously coated with 10 μg/ml (w/v) PDL. After purifying the cultures from microglia and OPCs (10–11 days in culture) the astrocyte medium was replaced by serum-free neuron medium composed of MEM (Thermo Fisher Scientific Inc.; Cat. No.: 31095029), 2% (v/v) B27 (Thermo Fisher Scientific Inc.; Cat. No.: 17504044), 0.1% (v/v) ovalbumin (Sigma-Aldrich; Cat. No.: A7641) 10 mM sodium pyruvate (Sigma-Aldrich; Cat. No.: S8636), and 0.1% (v/v) gentamicin (Sigma-Aldrich; Cat. No.: S9135) and conditioned for 24 h. Then, the supernatant was collected and stored at –80°C until the cytokine array analysis was performed. For the cytokine array 1 ml of the astrocyte supernatant was used for every experimental repetition. The chemiluminescence signals were measured with a chemiluminescence reader from Biostep and an exposure time of 2 min was set. The quantification of the signals was performed via ImageJ and the signal intensities were normalized against the internal positive controls of the array.

R&D Systems Proteome Profiler Mouse Cytokine Array Panel A consists of 40 capture antibodies spotted in duplicate on a nitrocellulose membrane. A cocktail of biotinylated detection antibodies was added to cell supernatants and these sample/antibody solutions were each added to and incubated on a separate membrane. Streptavidin-HRP and chemiluminescent reagents were added to the membranes and subsequently analyzed for light production at each spot via ChemiDoc MP Imaging System. Signal for each dot was calculated as integrated pixel density using Western Vision Quick Spots HLImage⁺⁺ software. Three independent experiments were performed, all with similar results.

50mg of snap frozen liver tissue was homogenized using a dounce homogenizer in 500μL PBS supplemented with protease inhibitor cocktail (Sigma-Aldrich). 1μL of Triton X-100 was added and lysate was frozen at -80°C for at least 30 minutes. Lysates were then thawed and centrifuged at 13,000g for 10 minutes to remove any debris. Array was performed on the lysate using the Proteome Profiler Mouse Cytokine Array, Panel A (ARY006; R&D Systems) according to provided instructions, detection was done with 2mL of chemiluminescence substrate, and exposure to photographic film (Kodak), quantification was performed using ImageJ [77 (link)].

‘Hot’ and ‘cold’ clones 2838c3 and 6694c2, were cultured (as explained above) and the supernatant fluid from these cells was collected in order to measure the cytokine and chemokine levels using the Proteome Profiler Mouse Cytokine Array Panel A (R&D systems, Minneapolis, USA, cat. No. ARY006). Everything was performed according to the manufacturer’s instructions and the membranes were measured using the IVIS Spectrum imager (Perkin Elmer). Experiments were repeated twice showing similar cytokine results.

Plasma cytokine protein content was measured using a Proteome Profiler: Mouse Cytokine Array Panel A (R&D Systems) to evaluate inflammatory mediators present in pooled plasma samples from the mice exposed to CCl₄ and euthanized 48 h later or in animals exposed to olive oil (control). In brief, pooled plasma samples (n = 6–8 individual mice per group) were applied to the provided membrane impregnated with capture antibodies. Streptavidin-HRP-conjugated secondary antibodies were then applied, and chemiluminescent technology was used to detect proteins captured by the array. All arrays were done on the same day with the same exposure time. NIH ImageJ was used to semi-quantify pixel density of resultant cytokine-positive areas recorded using radiographic film.

Kidneys were removed 1 or 2 days post-infection following PBS perfusion and homogenized on ice in 0.5 or 1 ml of PBS respectively. Chemokines and cytokines from homogenates and cell culture supernatants were analyzed according to manufacturer's instructions. Briefly, clarified samples were incubated with either BD cytometric bead array kits (IL-6, KC, MIP-1α, TNF, IL-1α), FlowCytomix Kits (IL-15/IL-15R, MCP-3 and IL-10), R&D Quantikine ELISA kit (IL-1β) or eBioscience Ready-Set-Go ELISA kit (GM-CSF). Bead based assays were assessed using a LSR Fortessa whilst ELISA samples were read at 450 nm with all concentrations determined relative to a standard curve.
For proteome profiling, kidneys were removed from naïve or 16 h post-infection mice following PBS perfusion and homogenized in 1 ml PBS with protease inhibitor cocktail (cOmplete Roche) with Triton ×100 added at a final concentration of 1% prior to a freeze thaw step. Samples were clarified prior to addition to the R&D Proteome profiler (Mouse cytokine array panel A) and developed as per manufacturer's instructions. Relative pixel density of each duplicate blot was measured using Image J software. The data are presented as fold change in signal from infected samples compared to naïve control samples.