We employed a glass chip-based multiplex sandwich ELISA system (QAH-CAA-4000; RayBiotech, Norcross, GA, U.S.A.) to measure the concentrations of 200 different human inflammatory mediators (Supplementary Table S1). This array platform was a combination of five forty-cytokine arrays and each antibody was printed in quadruplicate. Measurements were performed according to the recommended protocols from the manufacturer. Briefly, tissue samples were weighed and homogenized and then the supernatants were harvested. The protein concentration of each extract was determined and normalized to 500 μg/ml. The arrays were blocked with sample buffer and then incubated with samples or serial diluted cytokine standards overnight at 4°C. After multiple washes, the arrays were incubated with a cocktail of biotinylated antibodies. The arrays were then washed and incubated with Cy3 equivalent dye-conjugated streptavidin. The images were captured using a microarray scanner (InnoScan 300; Innopsys, Carbonne, France). The fluorescence intensity data were analyzed with the array-specific Q-Analyzer Software (RayBiotech). The results were expressed as picogram per milliliter (pg/ml).
Innoscan 300
Manufactured by Innopsys
Sourced in France
The InnoScan 300 is a high-performance, versatile microarray scanner designed for a wide range of applications. It features a dual-channel detection system, allowing for simultaneous scanning of two fluorescence channels. The scanner offers a large scan area, high resolution, and fast scanning speeds to meet the needs of various microarray-based experiments.
Automatically generated - may contain errors
Lab products found in correlation
Q analyzer software, by RayBiotech (1 mentions)
Well wash versa chip washer, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 goat anti human igg, by Thermo Fisher Scientific (1 mentions)
Wellwash versa, by Thermo Fisher Scientific (1 mentions)
Transwell permeable support, by Corning (1 mentions)
Human cytokine antibody array, by RayBiotech (1 mentions)
9 protocols using innoscan 300
1
Multiplex Sandwich ELISA for Inflammatory Mediators
2
Liver Tissue Scanning Protocol
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Thawed liver samples, as described above, were added to each well of the GSM-CAA-4000 chip (RayBiotech, Inc., Guangzhou) and incubated overnight. The samples were subjected to repeated cleaning with the Thermo Scientific Well wash Versa chip washer. Scanning signals were obtained with use of a laser scanner (InnoScan 300, Innopsys, Carbonne – France). Data analysis was performed using chip-specific data analysis software.
3
Protein Microarray for PvRBP1a Antigenicity
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Protein microarray was performed for evaluating total antigenicity using recombinant PvRBP1a-N and PvRBP1a-C. Three aminopropyl-coated slides were prepared as described previously [42 (link)]. The slides printed with optimised concentration of recombinant protein (PvRBP1a-N, 100 ng/μl and PvRBP1a-C, 50 ng/μl) to each spot were incubated for 2 hours at 37°C, and these slides were blocked with blocking buffer (5% BSA in PBS-T, 0.1% Tween 20) for 1 hour at 37°C. Healthy and vivax malaria patient sera were diluted in PBS-T to 1:25 ratio and treated on the chip for 1 hour at 37°C. The arrays were visualized with 10 ng/μl of Alexa Fluor 546 goat anti-human IgG (Invitrogen, Carlsbad, CA, USA) in PBS-T for 1 hour at 37°C and scanned using InnoScan 300 (INNOPSYS, France). The positive cut-off values calculated by negative control mean fluorescence intensity (MFI) plus two standard deviations.
Additionally, antigens were heat-treated at 80°C for 5 minutes to characterise the epitope. The linearized antigen also used for the microarray as described above.
Additionally, antigens were heat-treated at 80°C for 5 minutes to characterise the epitope. The linearized antigen also used for the microarray as described above.
4
Cytokine Detection in Mucosal and Serum Samples
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QAP-CYT-1 kits (Ray Biotech, Guangzhou, China) were used for cytokine detection and quantitation. Mucosal and serum samples were treated with 1× lysis buffer containing protease inhibitor cocktail, and the protein concentration of the lysate was determined using a BCA assay. For the detection of cytokines, samples were incubated on glass slides overnight at 4 °C, washed using a Thermo Scientific Well Wash Versa (Waltham, MA, USA), and incubation with Cy3 Equivalent Dye-streptavidin. After removal of unbound material by washing, an Inno Scan 300 (Innopsys, Parcd’ Activités Activestre, Carbonne, France) was used for fluorescence detection (scanning signals and Cy3 or green channels; excitation frequency = 532 nm).
5
Endothelial Cell Monolayer Permeability Assay
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In vitro endothelial cell monolayer permeability was assessed as previously described [28 (link)]. Briefly, HRECs were grown to confluence on gelatin-coated inserts of Transwell Permeable Supports (0.4 µm, Costar, Corning, NY, USA) and incubated at 37 °C for 6 days in low-glucose, high-glucose, or HGM conditions. The treated cells were then probed with 1 mg/mL 40-kDa FITC-dextran for 60 min, and the amount of FITC-dextran in the lower chambers of the culture wells was measured by a fluorescence scanner (InnoScan 300, Innopsys, Carbonne, France) using well-type arrays (n = 4), fabricated by mounting PDMS gaskets onto clean glass slides as previously described [29 (link)].
6
Cytokine Profiling with Microarray Assay
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The serum levels of various cytokines were evaluated using commercial assays (Human Inflammation Array 1, RayBiotech, USA) following the manufacturer’s instructions. We analyzed the following cytokines: IFN-γ, IL-1 α, IL-1β, IL-10, IL-13, IL-4, IL-6, IL-8, monocyte chemotactic protein (MCP)-1, and tumor necrosis factor alpha (TNF-α). An InnoScan 300 microarray scanner (Innopsys, France) was used to visualize signals. The RayBio Q-Analyzer Tool (QAH-INF-3-SW) was used to convert the data to concentrations.
7
Cytokine Profiling of LPS-Treated Cells
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The protein lysate samples of LPS-treated HIEC-6 cells were analyzed using the Human Cytokine Antibody Array 4000 (RayBio, GA, USA), according to the manufacturer’s instructions. Briefly, the array slide was incubated with 65 µL of LPS-treated HIEC-6 cell lysate overnight at 4 °C and equilibrated to room temperature on the following day. Then, the array slide was incubated for 2 h after extensive washing with an array-specific biotinylated antibody cocktail. The slide was incubated with Cy3-equivalent dye-conjugated streptavidin for 1 h. Finally, an InnoScan 300 microarray scanner (Innopsys, IL, USA) was used to obtain the images. To minimize false-positive hits, each sample was screened twice, and only the hits that appeared on both screenings were analyzed.
8
Tibetan and Han Cytokine Profiling
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Serum samples from four Tibetan, four Han at high-altitude and four Han at mid-altitude were utilized for analysis of the circulating cytokine profile using an antibody array (Human Cytokine Antibody Array, RayBiotech, Norcross, GA, USA) simultaneously measuring 440 cytokines via a sandwich method. Briefly, serum was added into array pools and incubated overnight at 4 °C. After washing, 440 biotin-conjugated antibody mix was added into the array pools for 2 hours incubation at room temperature. Subsequently, Cy3-conjugated streptavidin was added into the array pools and incubated for 2 hours. Finally, the arrays were scanned by a microarray scanner (InnoScan 300, Innopsys, France) for the fluorescent signal visualization.
9
Monkey Cytokine Array Analysis
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Plasma samples from three control and three monkeys injected with 320 EU IRV were used for monkey cytokine array analyses (Cat#: QAN-CYT-1-1) of eight cytokines (RayBiotech, Norcross, GA, USA), according to the manufacturer’s instructions. The fluorescent images were scanned by a microarray scanner (InnoScan 300; Innopsys, Parc d′Activites Activestre, France), and the data were analyzed with Mapix software. The cytokines were quantified according to the standard curve calibrated from the same array.
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