Human Cytokine 440 array of serum of preeclampsia patients and normal controls
Description
Cytokine family proteins may play an important role in the molecular mechanism of preeclampsia. In order to study the role and mechanism of cytokine family proteins in the pathogenesis of preeclampsia, we collected serum samples from four uncomplicated preeclampsia patients (Preeclampsia and No complications, PA936054, PA902074, PA932830, PA930563) and four normal controls (Normal Controls, NC919443, NC926142, NC918048, NC921466) and tested by Human Cytokine 440 array. As described in the document "GSH-CAA-440 English Protocol English operating instructions. pdf", after the serum samples were tested by multiple ELISA, the gray value of the chip was read and data analysis was performed to screen the cytokine family proteins differentially expressed in preeclampsia patients and normal controls.
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Steps to reproduce
1. Experimental materials and reagents 1.1 GSH-CAA-440 kit, the kit contains: The kit should be stored at -20℃. After starting to use, the slide chip, detection antibody mixture, Cy3-streptavidin should be stored at -20℃, and other reagents should be stored at 4℃ to avoid repeated freezing and thawing. A.Quantibody®Array Glass Chip B.Sample Diluent C.20X Wash Buffer I D.20X Wash Buffer II E.Detection antibody cocktail F.Cy3-conjugated Streptavidin G.Slide washer/Dryer H.Adhesive device sealer I.Operation Manual 1.2 Required materials and instruments other than the kit: • Plastic centrifuge tubes (2-5mL, 50mL) • Shaker • Plastic wrap •Aluminum foil • Double distilled water • InnoScan 300 Microarray Scanner fluorescence scanner • Thermo Scientific Wellwash Versa Microarray Washer 1.3 Sample: Human Serum X 16 2. Experimental steps 2.1 Complete drying of slide chips Take the slide chip out of the box, and after equilibrating at room temperature for 20-30min, open the packaging bag, peel off the seal, and then place the chip in a vacuum desiccator or dry at room temperature for 1-2 hours. 2.2 Chip operation process 2.2.1 Add 100 µL of sample diluent to each well, incubate for 1 h on a shaker at room temperature, and block the quantitative antibody chip. 2.2.2 Aspirate the buffer from each well, add 100µL of sample to the well, and incubate at 4℃ overnight (sample 2-fold dilution for loading). 2.2.3 Use the Thermo Scientific Wellwash Versa chip washer to wash the slides. It is divided into two steps. First, wash with 1× Wash Solution I, 250 µL of 1× Wash Solution I per well, wash 10 times, and shake for 10 s each time. Choose high shaking intensity and dilute 20× Wash Solution I with deionized water. Then use the 1× Washing Solution II channel for washing, 250 μL of 1× Washing Solution II per well, wash 6 times, shake for 10 s each time, choose a high vibration intensity, and dilute 20× Washing Solution II with deionized water. 2.2.4 Incubation of detection antibody mixture Centrifuge the detection antibody mixture vial, then add 1.4 ml of sample diluent, mix evenly, and centrifuge again quickly. Add 80 µL of detection antibody to each well and incubate for 2 hours on a RT shaker. 2.2.5 Cleaning, same as step 2.2.3 2.2.6 Incubation with Cy3-streptavidin Centrifuge the Cy3-streptavidin vial, then add 1.4 ml of sample diluent, mix well, and centrifuge again quickly. Add 80 µL of Cy3-streptavidin to each well, wrap the slides in aluminum foil and incubate in the dark for 1 hour on a RT shaker. 2.2.7 Cleaning, same as step 2.2.3 2.2.8 Fluorescence detection Scan the signal with a laser scanner such as InnoScan 300 using Cy3 or green channel (excitation frequency = 532 nm) 1) Instrument model: InnoScan 300 Microarray Scanner 2) Manufacturer: Innosys 3) Origin: Parc d'Activités Activestre; 31 390 Carbonne – France 4) Scanning parameters: WaveLengh: 532nm; Resolution: 10µm 2.2.9 The data analysis software of GSH-CAA-440 was used for data analysis.