Cytokine arrays were purchased from RayBiotech (Norcross, GA, USA). Human Cytokine Array C3 and Porcine Cytokine Array q1 were used to measure cytokines in culture supernatant. In summary, cells were serum starved for 16 hrs followed by 24 hrs of the indicated treatment. Conditioned media was collected and centrifuged at 200 xg for 10 min and cytokines involved in known inflammatory cascades in renal epithelial cells were profiled according to the manufacturer’s instructions. For these experiments, arrays were run with conditioned media from n = 3 pooled samples per array and n = 3 arrays per group for the human cytokine array and n=4 arrays per group for the porcine cytokine array. Signal intensity was measured for each spot and local background was subtracted for each array. Signal intensity on each array was normalized using the internal controls provided prior to calculating the mean values of the respective groups. Fold change was calculated by dividing the data from the treated group by the data from the control group.
Cytokine arrays
Manufactured by RayBiotech
Sourced in United States
Cytokine arrays are a type of laboratory equipment used to simultaneously detect and quantify multiple cytokines in a biological sample. They consist of an array of antibodies specific to various cytokines, which are immobilized on a solid support. The sample is incubated with the array, and the bound cytokines are detected and quantified using a labeled detection antibody or other detection method.
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6 protocols using cytokine arrays
1
Profiling Cytokine Secretion in Renal Cells
2
Cytokine Array of Serum-Starved Cells
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Cells were seeded on top of a thin layer of Matrigel as described and then serum starved overnight on day 7. Conditioned media were collected on day 8, and cell debris was removed by centrifugation. Cytokine arrays were performed according to the manufacturer's instructions (RayBiotech, Norcross, GA).
3
Cytokine Profiling of U87 Cells
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Cytokines secreted by U87MG and U87‐miR 302/367 cluster cells were detected in cytokine arrays (RayBiotech). The procedure was performed according to manufacturer's protocol. 1 × 106 cells were plated on 100 mm dishes in DMEM medium. The next day, the DMEM medium was discarded, cells were washed two times with PBS and cultured for 2 days in serum free DMEM medium. The medium containing the secreted cytokines was collected and briefly centrifuged. 1 ml of the supernatant was incubated with the human antibody array membranes at 4°C overnight, followed by membrane blocking. The membrane was incubated with biotin‐conjugated anti‐cytokines at room temperature for 2 hrs. Then 1:1,000 of diluted IRDYE 800CW streptavidin (LI‐COR Biosciences) was added followed by a washing step. Florescence signals on the membranes were detected and scanned by Odyssey Infrared Imaging System. The experiments were carried out in duplicate, and the Odyssey software was used to analyze the pixel density of each cytokine. Each of the cytokine pixel densities was normalized to positive and negative pixel densities.
4
Conditioned Medium Cytokine Analysis
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Conditioned medium (CM) was prepared by washing cells twice with 1× PBS, adding fresh DMEM with 0.2% FBS, incubating cells for an additional 24–36 h, and collecting the CM for further analysis. Individual cytokines secreted into the culture medium were measured using either human Quantikine ELISA kits (R&D Systems) or cytokine arrays (RayBiotech) (Supplemental Material ).
5
Lung Cytokine Expression Analysis
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Lung cytokine expression was determined using cytokine arrays purchased from Raybiotech (catalog # AAM-CYT-3). Mice were infected with SeV (500 pfu/gram body weight) for 3 or 7 days and the lungs were excised and homogenized in PBS supplemented with protease inhibitors. Cleared lung homogenate protein (500 μg) was used for cytokine determination. The membranes were developed and quantified using a BioRad Chemidoc XRS+ imager. The intensities were normalized to internal positive controls.
6
Detecting Cytokine Levels in Cell Supernatants
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