ET-1 in each culture supernatant was measured using cytokine-specific ELISA kits (BioSource and Endogen), in accordance with the manufacturers’ protocols.
Cytokine specific elisa kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
Cytokine-specific ELISA kits are quantitative immunoassay products used to measure the concentration of specific cytokines in biological samples. These kits employ the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify target cytokines.
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19 protocols using cytokine specific elisa kit
1
Quantification of ET-1 in Cell Cultures
2
Isolation and Analysis of CD8+ T Cells
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Total RNA was isolated from solid tumors or from CD8 þ T cells purified from solid tumors using the Tri-reagent (Invitrogen, Carlsbad, CA). CD8 þ T cells were positively selected using magneticactivated cell sorting (Miltenyi). Total RNA was reverse-transcribed with a first-strand cDNA synthesis kit for RT-PCR (PrimeScript RT Reagent Kit; Takara Bio), according to the manufacturer's instructions. The reverse-transcribed cDNA was then subjected to qPCR (SYBR Premix Ex Taq; Takara Bio, Shiga, Japan) and thermal cycling (Mx3000P Real-Time qPCR Systems; Stratagene, Santa Clara, CA). The reaction conditions were denaturation at 95 C for 30 seconds, followed by 40 cycles of denaturing at 95 C for 5 seconds, and annealing and extension at 60 C for 20 seconds. Levels of mRNA expression were estimated from the fluorescence intensity relative to b-actin. ELISA CD8 þ T cells were cocultured with B16 melanoma cells for 48 hours. IL-10 and IFN-g in each culture supernatant were measured using cytokine-specific ELISA kits (BioSource, Blue Bell, PA, and Endogen, Rockford, IL) according to the manufacturers' protocols.
3
Measuring IL-1β Secretion from Cardiomyocytes
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IL-1β proteins in the media from primary cardiomyocytes were measured by using cytokine-specific ELISA kits (eBiosciences Inc., CA, United States ).
4
Cytokine Quantification in Kidney Tissues
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TNF-α and IL-6 proteins in kidney tissues were determined by using cytokine-specific ELISA kits (eBiosciences Inc, CA, USA).
5
Cytokine Profiling of DC Supernatants
6
Quantifying IL-8 in Cell-Free Supernatants
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Cell-free supernatants were saved and aliquots were stored at −70 °C until use. The protein levels of IL-8 in medium were measured with commercially available cytokine specific ELISA kits (eBioscience, USA) according to the manufacturers’ recommendations.
7
Measuring TNF-α and IL-6 Proteins
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Tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) proteins in culture media and serum samples were determined by using cytokine‐specific ELISA kits (eBioscience, San Diego, CA). The amount of TNF‐α and IL‐6 in samples was normalized to total proteins.
8
T Cell Activation Assay with OVA Peptides
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Each single-cell suspension, isolated from the spleens of OVA-specific T cell receptor transgenic mice (OT-I and OT-II mice from C57BL/6 backgrounds) and BALB/C mice were labeled with bead-conjugated anti-CD4 and anti-CD8 mAbs (Miltenyi Biotec) and separated by sequential passaging using LS columns. Selected cells were stained with CFSE (5 μM, eBioscience) at 37°C and then washed with PBS (supplemented with 2% FBS) after 15 min. Next, in the presence or absence of OVA-specific OVA323–339 or OVA257–264 treatments, the cells (non-, LPS-, MAH-, MAH/LPS-treated DCs) were co-cultured with CFSE-labeled T cells (from naïve BALB/C, OT-I, or OT-II mice) at a DC to a T cell ratio of 0.5:1. After 3 days of co-culture, cells were harvested and stained with anti-CD4 and anti-CD8 mAbs (eBioscience). Data were collected on LSRII and analyzed using the FlowJo software. Cytokines, including IFN-γ and IL-2, were analyzed from the culture supernatants using cytokine-specific ELISA kits (eBioscience), following the manufacturer’s instructions.
9
Cytokine Profiling in Mice
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The concentrations of serum IL-1β, IL-6, IL-10, IL-23, TNFα, and IL-17A in individual mice were determined using the cytokine-specific ELISA kits (eBioscience, San Diego, CA, USA), according to the manufacturer’s instructions.
10
Quantification of IL-18 and IL-1β in H9c2 Cells
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IL-18 and IL-1β protein levels in H9c2 cell media were determined using cytokine-specific ELISA kits (eBiosciences Inc, CA).
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