A Human Cytokine Array Panel A (ARY005; R&D Systems, Minneapolis, MN, USA) and a Human Chemokine Array Kit (ARY017; R&D Systems, Minneapolis, MN, USA) were used to profile 36 cytokines and 31 chemokines, respectively, in the supernatants from cell cultures. The array membranes were incubated in blocking buffer for 1 h at room temperature. Subsequently, 1.5 mL of the sample/antibody mixture was added per well, followed by incubation overnight at 2–8 °C on a rocking platform shaker. The membranes were washed three times in wash buffer (ARY005; R&D Systems, Minneapolis, MN, USA) at room temperature. Next, streptavidin-HRP in array buffer was added, and the membranes were incubated for 30 min at room temperature. The membranes were washed again, followed by the addition of Chemi Reagent Mix (R&D Systems, Minneapolis, MN, USA) for 1 min. The membranes were visualized using a PXi multi-application gel imaging system (Syngene, Cambridge, UK).
Ary005
Manufactured by R&D Systems
Sourced in United States
ARY005 is a multiplex enzyme-linked immunosorbent assay (ELISA) kit designed to simultaneously detect and quantify multiple analytes in a single sample. The kit utilizes magnetic beads coated with capture antibodies specific to the target analytes. The captured analytes are then detected using biotinylated detection antibodies and a streptavidin-conjugated reporter system.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd68 antibody, by Agilent Technologies (2 mentions)
Vectastain kit, by Agilent Technologies (2 mentions)
Human cytokine array panel a, by R&D Systems (1 mentions)
Chemi reagent mix, by R&D Systems (1 mentions)
Pxi multi application gel imaging system, by Syngene (1 mentions)
Human chemokine array kit, by R&D Systems (1 mentions)
Drg00, by R&D Systems (1 mentions)
K 1200, by Echelon Biosciences (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
8 protocols using ary005
1
Cytokine and Chemokine Profiling in Cell Cultures
2
Profiling Cytokine Secretion via ELISA
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A human cytokine array kit was purchased from R&D Systems (Catalog # ARY005, Human Cytokine 37-membrane kit array) and utilized following the protocol provided by the manufacturer. Capture antibodies have been spotted in duplicate on nitrocellulose membranes. Cell culture supernatants are mixed with a cocktail of biotinylated detection antibodies. The sample/antibody mixture is then incubated with the array. Any cytokine/detection antibody complex present is bound by its cognate immobilized capture antibody on the membrane. Streptavidin-Horseradish Peroxidase and chemiluminescent detection reagents are added. A signal is produced, proportional with the amount of cytokine bound. Chemiluminescence is detected in the same manner as a Western blot and the resulting cytokine release profiles were quantified with ImageJ.
3
Evaluating Protein Expression and Macrophage Infiltration in CMT1A
4
Cytokine Profiling of Apoptotic Cell-Stimulated Macrophages
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Ben-Men-I cells were seeded at a concentration of 6 × 104/mL in DMEM medium and incubated with unlabeled apoptotic cells. After 24 h, the culture supernatants were harvested, centrifuged at 1,000 rpm for 5 min, and assayed for IL-6, IL-8 (Orgenium Laboratories), IL-1RA (R&D, DRA00B), IL-16 (RayBiotech, ELH-IL16-001), MIF (RayBiotech, ELH-MIF-001), or CXCL1 (R&D, DRG00) by ELISA. Cytokines present in apoptotic body preparations were measured and background subtracted. The assays were performed according to the manufacturers’ instructions. Absorbance was read on a Spectramax GEMINI XS microplate reader (450 nm). For multiplexed detection of cytokines/chemokines, the human cytokine array panel A (R&D, ARY005) was used according to manufacturers’ recommendations.
5
Evaluating Protein Expression and Macrophage Infiltration in CMT1A
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To evaluate CXCL1 and MCP-1 protein expression, de-identified sural nerve biopsies from two patients with genetically proven CMT1A (two women, ages 26 and 50) and two histologically normal samples from non-CMT1A patients (two women, ages 55 and 79) were obtained from the Johns Hopkins Neuromuscular Histopathology Laboratory under appropriate Institutional Review Board (IRB) supervision with informed consent. Lysates were obtained by sonicating nerve biopsies in RIPA buffer, and equal amounts of protein were then used for secreted protein expression profiling (R&D Systems, ARY005).
To evaluate the presence of CD68+ monocytes/macrophages, a diagnostic biopsy of the intercostal nerve was done in a 28 year old female patient with genetically confirmed CMT1A to evaluate for enlargement of her nerves and to rule out Schwannomatosis (under same IRB as above). She was diagnosed with CMT1A only a year prior to biopsy when she presented with pain and enlarged nerve roots and lumbosacral and brachial plexi. The biopsy showed classic features of an inherited demyelinating neuropathy with axonal loss and many mature onion bulbs. CD-68 immunostaining was performed in paraffin embedded sections with anti-CD68 antibody (DAKO cat # M071801–5) using standard immunohistochemical methods (Vectastain kit).
To evaluate the presence of CD68+ monocytes/macrophages, a diagnostic biopsy of the intercostal nerve was done in a 28 year old female patient with genetically confirmed CMT1A to evaluate for enlargement of her nerves and to rule out Schwannomatosis (under same IRB as above). She was diagnosed with CMT1A only a year prior to biopsy when she presented with pain and enlarged nerve roots and lumbosacral and brachial plexi. The biopsy showed classic features of an inherited demyelinating neuropathy with axonal loss and many mature onion bulbs. CD-68 immunostaining was performed in paraffin embedded sections with anti-CD68 antibody (DAKO cat # M071801–5) using standard immunohistochemical methods (Vectastain kit).
6
Cytokine Profiling of HaCaT Cells
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HaCaT‐OE and HaCaT‐VE cells were cultured normally according to the above conditions, and the supernatants were subsequently harvested after 48 h. Human cytokine array was used to identify the supernatants (R&D Systems, ARY005).
7
Cytokine Profiling of Transmigrated Cells
8
Quantification of Hyaluronan Dynamics
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Hyaluronan in the culture media and pericellular matrix released by the trypsin-EDTA treatment (see Supplementary Materials) were analyzed with a sandwich-type (Hiltunen et al., 2002) or competitive ELSA (K-1200; Echelon Biosciences, Salt Lake City, UT).
RNA extraction, cDNA synthesis, qPCR, SDS-PAGE, and Western blotting were performed as described previously (Pasonen-Seppa ¨nen et al., 2012b; Takabe et al., 2015) and in the Supplementary Materials and Supplementary Table S1.
The culture media were screened for cytokines with a commercial array (ARY005; R&D Systems, Minneapolis, MN). The intensities of the spots, imaged with a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA), were quantified with the ImageJ 64 software (National Institutes of Health, Bethesda, MD).
The staining for hyaluronan and the microscopy were performed as described previously (Rilla et al., 2008) and in the Supplementary Materials.
RNA extraction, cDNA synthesis, qPCR, SDS-PAGE, and Western blotting were performed as described previously (Pasonen-Seppa ¨nen et al., 2012b; Takabe et al., 2015) and in the Supplementary Materials and Supplementary Table S1.
The culture media were screened for cytokines with a commercial array (ARY005; R&D Systems, Minneapolis, MN). The intensities of the spots, imaged with a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA), were quantified with the ImageJ 64 software (National Institutes of Health, Bethesda, MD).
The staining for hyaluronan and the microscopy were performed as described previously (Rilla et al., 2008) and in the Supplementary Materials.
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