Splenocytes from individual mice were cultured in RPMI 1640 medium containing 10% of fetal bovine serum, L-glutamine (2 mM), penicillin (50 U/mL), and streptomycin (50 µg/mL) (all from Biowest SAS, Nuaillé, France) at a cellular density of 4×106 cells/mL in 24-well plates. They were stimulated in triplicate with concanavalin A (2.5 µg/mL), OVA-LPS, or OVA-LPS-free (200 µg/mL) and culture medium as a control. Cells were maintained for 72 hours at 37℃ in 5% CO2. Afterward, plates were centrifuged and culture supernatants collected and stored at -80℃ until analysis. IFN-γ, TFN-α, IL-5, IL-10, and IL-13 were quantified using cytokine ELISA kits (eBioscience) as outlined by the manufacturers. The results were expressed in pg/mL as sample means for test replicates.
Cytokine elisa kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Cytokine ELISA kits are a type of laboratory equipment used for the quantitative measurement of cytokines in biological samples. These kits utilize the Enzyme-Linked Immunosorbent Assay (ELISA) technique to detect and quantify specific cytokines present in the sample. The core function of these kits is to provide a standardized and reliable method for the analysis of cytokine levels, which are important biomarkers in various fields of biological and medical research.
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Lab products found in correlation
Tnf α, by Thermo Fisher Scientific (2 mentions)
Anti cd28, by BD (2 mentions)
Anti cd3, by BD (2 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Biowest (1 mentions)
Streptomycin, by Biowest (1 mentions)
L glutamine, by Biowest (1 mentions)
Human factor x, by Enzyme Research (1 mentions)
Mouse tat complex elisa kit, by Assaypro (1 mentions)
Igg2a, by Southern Biotech (1 mentions)
96 well microplate, by Merck Group (1 mentions)
Cytokine enzyme linked immunosorbent assay elisa kits, by Thermo Fisher Scientific (1 mentions)
Phospho nf κb p65, by Abcam (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Wortmannin, by Merck Group (1 mentions)
Antibody to β actin, by Merck Group (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Hbeag, by ProSpec (1 mentions)
31 protocols using cytokine elisa kit
1
Splenocyte Cytokine Production Assay
2
Investigating Mycobacterium tuberculosis Pathogenesis
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Gamma-irradiated H37Rv (γ-Mtb) and Mycobacterium tuberculosis (M.tb) H37Rv (27294) were obtained from ATCC (Rockville, MD). Recombinant human factor VIIa (FVIIa) was from Novo Nordisk (Gentofte, Denmark). Human factor X was purchased from Enzyme Research Laboratories (South Bend, IN). Chromogenic substrate Chromogenix S-2765 was from DiaPharma (West Chester, OH). Cytokine ELISA kits were from eBioscience (San Diego, CA). Mouse TAT-complex ELISA kit was from Assaypro (St. Charles, MO). Mouse macrophage marker antibody F4/80 (SP115) was from Novus Biologicals (Littleton, CO) and neutrophil marker Ly-6G (IA8) was from BD Pharmingen (San Diego, CA). Preparation and characterization of monospecific polyclonal antibodies against human TF were described earlier [36] (link). Rabbit polyclonal anti-mouse TF antibody was kindly provided by Lars C. Petersen, Novo Nordisk, Denmark.
3
ELISA-based Measurement of Virus-specific Antibodies and Cytokines
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Virus-specific antibodies were determined in samples by enzyme-linked immunosorbent assay (ELISA) as previously described [23 (link), 31 (link)]. Briefly, FI-RSV (4 μg/ml) was used as a coating antigen. The antibody responses were detected using the secondary antibodies of horse radish peroxidase-conjugated goat anti-mouse IgG, IgG1, and IgG2a (Southern Biotechnology). Antibody concentrations were quantified using the standard curve for each IgG isotype antibodies. The levels of interleukin-4 (IL-4) and IL-5, IL-6, interferon (IFN)-γ and tumor necrosis factor (TNF)-α (eBioscience, SanDiego, CA) in lung extracts and bronchoalveolar lavage fluid (BALF) homogenates were measured using cytokine ELISA kits (eBioscience, SanDiego, CA).
4
Splenocyte Activation and IFN-γ Measurement
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Red blood cell-depleted splenocytes were cultured at 4 × 105 cells/well in a total volume of 200 μl. Splenocytes were stimulated with 1 μg/ml anti-CD3 and 1 μg/ml anti-CD28. Cell-free supernatants were collected, and subjected to assays to measure IFN-γ production using cytokine ELISA kits (eBioscience, Frankfurt, Germany).
5
Cytokine Analysis in Cell Cultures
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For cytokine analysis, cell cultures were prepared as described above, and the supernatants were collected at the indicated times and stored at -20°C. The supernatant concentrations of IFN-γ and TNF-ɑ were determined using cytokine ELISA kits (eBioscience). The results were analyzed spectrophotometrically at 450 nm using an ELISA microplate reader.
6
Cytokine Production After Viral Immunization
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To detect the cytokines produced after immunization, splenocytes were isolated from each immunized mouse, mixed, and cultured in 96-well microplates (Millipore, Billerica, MA, USA) at a density of 2 × 104 cells/well with 100 μl of RPMI 1640 containing 10% FBS and 10 μg of inactivated EV71 virus or gag-peptide complex (Huaxia Yuanyang Tech, Beijing, China) for stimulation. After incubation at 37°C for 48 h, the plate was centrifuged at 1000 rpm for 5 min, and the cell culture supernatant was collected carefully and assayed for interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ, using cytokine ELISA kits (eBioscience).
7
Cytokine Secretion Profiling
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After 18 h of co-culture cells at a 1:1 E:T ratio, the supernatants were collected, and the levels of interferon γ (IFN-γ) and interleukin-2 (IL-2) in the supernatant were detected using cytokine ELISA kits (e-Bioscience, San Diego, CA, USA) according to the manufacturer’s instructions.
8
Cytokine ELISA for IL-10 and IFN-γ
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IL-10 levels in the serum and in the cell-free supernatants were determined by the corresponding cytokine ELISA kits (eBioscience, San Diego, CA, USA), according to the corresponding manufacturer’s instructions. Red blood cell (RBC)-depleted splenocytes were cultured at 1×106 cells per well with or without KCs in a total volume of 200 µL at a ratio of 2:1 (splenocytes: KCs). Splenocytes were stimulated with anti-CD3 (1 µg/mL) and anti-CD28 (1 µg/mL) (BD Bioscience, Franklin Lakes, NJ, USA). Interferon (IFN)-γ levels in the cell-free supernatants were determined by the corresponding cytokine enzyme-linked immunosorbent assay (ELISA) kits (eBioscience), according to the corresponding manufacturer’s instructions.
9
TLR5 Signaling Pathway Activation
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Purified flagellin from S.typhimurium ligand (ultrapure) TLR5 was purchased from InvivoGen (San Diego, CA, USA). Wortmannin was purchased from Sigma Chemical (St Louis, MO, USA). Rapamycin was purchased from Gene Operation (Ann Arbor, Michigan, USA). LY294002 was purchased from Cell Signaling (Beverley, MA, USA). Antibody to β-actin was purchased from Sigma Chemical. Antibodies to phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), phospho-Akt (Ser473), Akt, phospho-S6 (Ser240/244), S6, phospho-4EBP1 (Thr37/46), IκBα, NF-κB p65, phospho-STAT3 (Tyr705), and STAT3 were purchased from Cell Signaling (Beverley, MA, USA). Antibodies to 4EBP1, phospho-mTOR (Ser2448), mTOR, TLR5, TLR4 and phospho-NF-κB p65 (Ser536) were purchased from Abcam (Cambridge, UK). Cytokine ELISA kits were obtained from eBioscience (San Diego, CA, USA).
10
Cytokine Regulation in NPC Cells
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Cytokines, including IL-1β, IL-6 and GM-CSF, from NPC cell lines or NPC-LMP1 cell lines were assessed after treatment with siGLUT1, siNLPR3, 2-DG or the caspase-1 inhibitor VX765 for 24 h. Supernatants were collected, and IL-1β, IL-6 and GM-CSF were measured using cytokine ELISA kits (eBioscience) following the manufacturer’s instructions.
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