Culture supernatants of differentially stimulated BMDCs were collected and stored at −70 °C for analysis of cytokine and NO production. Measurement of IL-12p40 levels was carried out as detailed in the instructions provided with cytokine ELISA kit (BD Biosciences). The accumulation of NO in the stimulated culture supernatants were measured as described previously58 (link). Briefly, 50 μl of the supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylene diamine hydrochloride in 50% H3PO4) and incubated at room temperature for 10 min. Absorbance was then measured at 540 nm in a plate reader (Thermo, Multiskan EX).
Cytokine elisa kit
Manufactured by BD
Sourced in United States
Cytokine ELISA kits are laboratory equipment designed to quantify the presence and concentration of specific cytokines in biological samples. These kits utilize the enzyme-linked immunosorbent assay (ELISA) technique to detect and measure target cytokines. The core function of these kits is to provide researchers and scientists with a reliable, standardized method for analyzing cytokine levels in various sample types.
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Lab products found in correlation
Multiskan ex, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
24 well tissue culture plate, by Corning (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Apoptosis detection kit, by Merck Group (1 mentions)
Cdna synthesis kit, by Roche (1 mentions)
Lactate dehydrogenase ldh assay kit, by Merck Group (1 mentions)
Trisodium citrate, by Merck Group (1 mentions)
Lipoic acid, by Merck Group (1 mentions)
Microtainer sst tube, by BD (1 mentions)
Cytometric bead array flex set system, by BD (1 mentions)
Luminex instrument, by Merck Group (1 mentions)
10 protocols using cytokine elisa kit
1
Quantifying Cytokine and Nitric Oxide Levels
2
Cytokine Profiling of Bioactive Silver Nanoparticles
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THP-1 cells were prepared as described above. After 24 h of incubation, the adhered cells were treated with bio-AgNPs and bio-AgNP CS/P407 hydrogel at concentrations ranging from 8−64 μg/mL. The final volume of each well was 300 μL, and the positive control was 1 μg/mL of LPS from E. coli. The plates were incubated for 24, 48, and 72 h at 37°C supplemented with 5% CO2. The supernatant was transferred to another 96-well plate and placed at −20°C until testing.
The levels of cytokines, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin 10 (IL-10) were estimated by cytokine ELISA kit from B&D according to the manufacturer’s instructions. After that, a solution of the developer TMB (100 μL) was added to the plates, and the reaction was stopped with 50 μL of 1M H2SO4. The plates were read at 450 nm using a microplate reader (Epoch, BioTek).
The levels of cytokines, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin 10 (IL-10) were estimated by cytokine ELISA kit from B&D according to the manufacturer’s instructions. After that, a solution of the developer TMB (100 μL) was added to the plates, and the reaction was stopped with 50 μL of 1M H2SO4. The plates were read at 450 nm using a microplate reader (Epoch, BioTek).
3
Splenocyte Cytokine Profiling after Immunization
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Four weeks after the last immunization, the mice were killed by cervical dislocation, and the spleens were aseptically removed from the mice. The spleens were ground and passed through a cell strainer. The cell suspensions were concentrated by centrifugation at 1000 r/min for 5 min, and the erythrocytes were lysed using ACK lysis buffer. The erythrocytes were removed, and the splenocytes were washed twice with RPMI-1640 (Gibco, Waltham, MA, USA) medium and then diluted to 1 × 106 cells/mL in RPMI-1640 medium supplemented with 10% FBS and 100 U/mL penicillin–streptomycin. Next, 0.5 mL of splenocytes (1 × 106) were seeded in duplicate in 24-well tissue culture plates (Corning, New York, NY, USA); the cells were stimulated with 0.5 mL of purified Rv2351c protein (5 μg/mL) and Ag85B protein (5 μg/mL) and cells that were stimulated with phytohemagglutinin (1 μg/mL) and AIV medium served as the positive and negative controls, respectively, and were grown at 37 °C with 5% CO2 for 72 h. The culture supernatants were collected, and the concentrations of IFN-γ, IL-2 and IL-4 in the supernatants were determined using the cytokine ELISA kit (BD Biosciences, NJ, USA).
4
Cytotoxicity and Immunomodulatory Assays
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M-199, RPMI-1640, and yeast peptone dextrose medium, HAuCl4•3H2O, Giemsa dye, Trypan blue, MTT, 2′,7′-dichlorodihydrofluorescein diacetate dye, N-acetyl l-cysteine (NAC), diphenyleneiodonium chloride, proteinase K, ergosterol (ERG), diphenyl hexatriene (DPH), RNase A, Griess reagent, 2,4-dinitrophenylhydrazine, TRIZOL, nitro blue tetrazolium, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), lipoic acid, sulfo-N-hydroxy succinamide (NHS), AmB as Fungizone, the Glutathione Colorimetric Assay Kit, the lactate dehydrogenase (LDH) assay kit, the Apoptosis Detection Kit, and all solvents were from Sigma-Aldrich Co. (St Louis, MO, USA). Trisodium citrate was from Merck (USA). The QIAamp DNA Mini kit and RNeasy Mini Kit were from Qiagen (NV, Venlo, the Netherlands). The cDNA synthesis kit was from Hoffmann-La Roche (Basel, Switzerland). The cytokine ELISA kit was obtained from BD Biosciences (San Jose, CA, USA) for mouse interferon (IFN)-γ, IL-12, and IL-10, respectively. The RAW 264.1 THP-1 cell line was obtained commercially from the National Cell Repository, NCCS, Pune, India. For the hemolysis assay, blood samples from healthy volunteers were collected based on ethical approval from RMRIMS-Patna (Reference no. 21/RMRI/EC/2017) and the donor’s written informed consent.
5
Serum Cytokine and Clinical Chemistry Analysis
6
Cytokine Response Evaluation in Mice
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Mice were euthanized 4 weeks after the last immunization and lymph node and spleen cells were isolated as described previously [20 (link)]. Cells from lymph nodes of mice from the same group were pooled before in vitro stimulation. For stimulation assays, cells from individual spleens or from pooled lymph nodes were dispensed at 2 × 105 cell/100 μL media/well in 96-well flat bottomed tissue culture plates (Sarstedt, USA) and incubated with 2 or 10 μg/mL of recombinant protein in complete medium (RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, 25 mM HEPES, penicillin (100 U/ml) plus streptomycin (100 lg/mL), and 50 μM β-Mercaptoethanol) for 72 hr at 37°C in a humidified incubator with 5% CO2. Cells were also incubated with 5 μg/ml concanavalin A (ConA) or with medium alone as positive and negative control, respectively. Culture supernatants were collected and cytokine production was measured using cytokine ELISA kits as per the manufacturer's instructions (BD Bioscience, ON, Canada) as described previously [20 (link)]. The amount of IFN-γ and IL-10 produced by lymph node or spleen cells was expressed as ng/ml. In addition, the production of IL-4 was measured in spleen cells and was expressed as pg/mL.
7
Serological and Cytokine Profiling of Toxoplasmosis
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T. gondii-specific antibody and cytokine levels were determined by ELISA as reported previously [18 (link),26 (link)]. Diluted immune sera (1:100), urine (1:3), vaginal samples (1:3), fecal supernatants (1:3), intestine samples (1:100), and brain supernatants (undiluted) were used as primary antibodies. Briefly, 96-well plates were coated with T. gondii RH (2 μg/mL) and blocked prior to incubation with the primary antibodies listed above. Anti-mouse IgG and IgA secondary antibodies conjugated with horseradish peroxidase (HRP) were used to detect the antigen-specific responses. The production of pro-inflammatory cytokines interferon-gamma (IFN-γ) and interleukin-6 (IL-6) were measured from the brain supernatants using the cytokine ELISA kits (BD Biosciences, San Jose, CA, USA).
8
Quantify IL-1β in Kidney Tissues
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After lysing kidney tissues, supernatants were collected from each group to detect the level of IL-1β using cytokine ELISA kits (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions.
9
Peritoneal Macrophage Cytokine Assay
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Peritoneal Mϕs were stimulated with 100 ng/mL LPS for 3 h and cell-free supernatants collected. IL-10 and IL-12p40 production was assayed using cytokine ELISA kits (BD Biosciences), according to the manufacturer's instructions.
10
Cytokine and Chemokine Profiling
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