The concentrations of human IFN-β (VeriKine™ Human IFN β ELISA; PBL Assay Science, Piscataway, NJ), IL-6 (Human IL-6 ELISA MAX™ Deluxe, Biolegend, San Diego, CA), IL-7 (Human IL-7 Quantikine HS ELISA Kit; R&D systems, Minneapolis, MN), and IL-15 (Human IL-15 Quantikine ELISA Kit; R&D systems) in conditioned media from human MSCs were measured in accordance with the manufacturer's protocols. All samples were examined in triplicate for each experiment.
Human il 15 quantikine elisa kit
Manufactured by R&D Systems
Sourced in United States
The Human IL-15 Quantikine ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay designed to measure human interleukin-15 levels in cell culture supernatants, serum, and plasma. It uses a microplate pre-coated with a monoclonal antibody specific for human IL-15.
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Lab products found in correlation
Human il 6 elisa max deluxe, by BioLegend (1 mentions)
Human il 7 quantikine hs elisa kit, by R&D Systems (1 mentions)
Human il 13 quantikine elisa kit, by R&D Systems (1 mentions)
Immunocult human cd3 cd28 cd2 t cell activator, by STEMCELL (1 mentions)
Safire microplate reader, by Tecan (1 mentions)
Powergen 700, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Serum separator tube, by BD (1 mentions)
Trypan blue exclusion assay, by Lonza (1 mentions)
Ab serum, by Valley Biomedical (1 mentions)
Aim 5, by Thermo Fisher Scientific (1 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
Flow cytometry, by BD (1 mentions)
Lipopolysaccharide, by Merck Group (1 mentions)
Cytomegalovirus igg elisa kit, by Abnova (1 mentions)
12 protocols using human il 15 quantikine elisa kit
1
Quantifying Cytokine Secretion in Human MSCs
2
Quantitative Measurement of IL-15
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For the measurement of IL-15 plasma levels, the Human IL-15 Quantikine ELISA Kit (R&D Systems) was used, following manufacturer’s recommendations. IL-15 plasma levels were measured in S1, S2, S3 and S4 patients´ samples. The optical density was determined using Varioskan Flash fluorimeter (Thermo Fisher Scientific) set to 450nm.
3
Serum Cytokine Profiling by CBA and ELISA
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The Cytometric Bead Array technique was used to detect the level of serum interleukin 15 (IL-15), IL-2, IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) (Human IL15/Th1/Th2 CBA kit, BD Bioscience, Franklin Lakes, NJ, USA). Serum IL-15 was confirmed by a separate enzyme-linked immunosorbent assay (ELISA) (Human IL-15 Quantikine ELISA Kit, R&D system, Minneapolis, MN, USA).
4
Quantification of IL-15 and IL-13 Secretion
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IL-15 production was measured using a Human IL-15 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. IL-13 production was analyzed using Human IL-13 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA). For measuring IL-15 secretion upon T-cell activation, 5.0 × 105 T cells were washed with PBS, resuspended in 275 µL of T-cell media (RPMI, 10% FBS, and 1% GlutaMAX) without cytokines and plated in 96-well, V-shaped plates. Cells were then activated with Immunocult™ Human CD3/CD28/CD2 T Cell Activator (StemCell Technologies, Vancouver, Canada) following the manufacturer’s protocol and incubated at 37 °C. After 24 h, 250 µL of media was collected for IL-15 ELISA from the wells and stored at −80 °C.
5
IL-15 Secretion in mACE2-CAR_sIL15 NK Cells
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Supernatants were harvested after mACE2-CAR_sIL15 NK and control NK cells were co-cultured without or with A549-spike cells for 72 h. IL-15 concentrations were measured with the human IL-15 Quantikine ELISA kit (Cat# S1500, R&D) following the manufacturer’s instructions. Each experiment was performed in triplicate.
6
Quantification of IL-15 in Cell Culture Supernatants
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Cells were seeded at 0.01 × 106 cells/ml in T25 flasks. After 5 days, the cell culture supernatant was collected for IL-15 measurement using the Human IL-15 Quantikine ELISA Kit (R&D Systems) following manufacturer's protocols. The absorbance was measured at 450 nm (reference set at 540 nm) using a Tecan Safire Microplate Reader (Tecan). A standard curve (constructed from standards provided in the kit) was used to determine the concentration of IL-15. Reported concentrations represent the mean ± SD of three replicate samples.
7
Quantifying Tumor Cytokine Profiles
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Tumors were collected from humanely euthanized mice, frozen immediately on dry ice and stored at -80°C. Blood, was collected from mice under mild anesthesia via retroorbital bleeding into serum separator tubes (BD). Serum was collected after centrifugation (5 minutes at 1000 rpm) at 4°C and stored at -20°C until analyzed. To measure cytokine levels within the tumor tissue, samples were thawed, weighed, and diluted in 500 pl-800 pl of PBS with protease inhibitor cocktail (Roche, Brandford, CT) dependent on sample size. Tissue samples were kept on ice and homogenized using a PowerGen 700 (Fisher Scientific, Pittsburg, PA). The homogenate was centrifuged at 1000 rpm for 5 minutes at 4°C. Expression levels of IL-15 were measured using a human IL-15 Quantikine ELISA kit (R&D Systems, Minneapolis, MN) per the manufacturer’s instructions. hIL-15 levels were calculated as pg/ml of tumor homogenate and normalized to tumor weight. The data is expressed as pg/0.1 g of tumor.
8
Evaluating CAR-NK Cell Cytokine Production and Recovery
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CAR-NK cells or CAR/IL15-NK cells (4 × 106/mL) were cultured with or without HCT-116 cells (E:T = 5:1) in 24-well plates (Thermo Fisher Scientific) for either 3 h or 24 h. The supernatant was collected for measuring of IL-15 production with the Human IL-15 Quantikine ELISA Kit (R&D System, Minneapolis, MN) following the manufacturer's protocol.
To test NK cell recovery in vitro, CAR-NK cells or CAR/IL15-NK cells (1 × 106/mL) were seeded into the 24-well plates (Thermo Fisher Scientific) with AIM-V (Invitrogen) supplemented with 5% AB serum (Valley Biomedical) and cultured with low concentration (10 IU/mL) of IL-2 or without IL-2 for 7 days. The IL-2 was replenished every 2 to 3 days. The viable cell number was determined by trypan blue exclusion assay (Lonza) and the CAR percentage was analyzed by flow cytometry (BD Biosciences).
To test NK cell recovery in vitro, CAR-NK cells or CAR/IL15-NK cells (1 × 106/mL) were seeded into the 24-well plates (Thermo Fisher Scientific) with AIM-V (Invitrogen) supplemented with 5% AB serum (Valley Biomedical) and cultured with low concentration (10 IU/mL) of IL-2 or without IL-2 for 7 days. The IL-2 was replenished every 2 to 3 days. The viable cell number was determined by trypan blue exclusion assay (Lonza) and the CAR percentage was analyzed by flow cytometry (BD Biosciences).
9
Quantifying human IL-15 in mice
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The Human IL-15 Quantikine ELISA Kit (Cat#D1500 R&D Systems, Minneapolis, MN, USA) was used to quantify levels of human IL-15 in the sera of Hu-NSG-Tg(IL-15) and humanized NSG mice according to the manufacturer’s guidelines, and optical density was determined using the SpectraMax® iD3 microplate reader set to 450 nm.
10
Measuring Human IL-15 in Mice
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IL15WT and IL15KI mice were injected with lipopolysaccharide (Sigma-Aldrich) at a dose of 0.4 mg/kg. Human IL-15 protein concentration was measured in the plasma 6 hours later using a human IL-15 Quantikine ELISA kit (R&D Systems).
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