Levels of proinflammatory cytokines, IL-1β, IL-6, IL-18, tumor necrosis factor alpha (TNFα), and high mobility group box 1 (HMGB1), were determined in cell culture medium at multiple time points post asbestos exposure (0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post asbestos) using enzyme-linked immunosorbent assays (ELISA). Samples were run undiluted in triplicate, and assays were performed according to manufacturer's instructions. Levels of IL-1β, IL-6, IL-18, and TNFα are reported as picograms per milliliter (pg/ml) of culture medium, and levels of HMGB1 released into the culture medium are reported as nanograms per milliliter (ng/ml). ELISA kits (TNFα and IL-1β) were purchased from BD biosciences (San Jose, CA, USA), MBL International (Woburn, MA, USA) (mouse IL-18 ELISA Kit), R&D systems (Minneapolis, MN, USA) (mouse IL-6 Quantikine ELISA Kit), and Chondrex Inc. (Redmond, WA, USA) (HMGB1 Detection Kit).
Mouse il 18 elisa kit
Manufactured by R&D Systems
Sourced in United States
The Mouse IL-18 ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of mouse Interleukin-18 (IL-18) levels in cell culture supernatants, cell lysates, serum, and plasma.
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Lab products found in correlation
Mouse il 6 quantikine elisa kit, by R&D Systems (2 mentions)
Mouse il 1β elisa kit, by R&D Systems (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Hmgb1 detection kit, by Chondrex (1 mentions)
Mouse il 6 quantikine elisa kit, by Chondrex (1 mentions)
Luminex 200, by Bio-Rad (1 mentions)
Bio plex mouse cytokine 23 plex panel kit, by Bio-Rad (1 mentions)
Bio plex manager software, by Bio-Rad (1 mentions)
Myricitrin, by Merck Group (1 mentions)
Apigenin, by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Lactate dehydrogenase ldh cytotoxicity detection kit, by Takara Bio (1 mentions)
Tricetin, by Merck Group (1 mentions)
Luteolin, by Merck Group (1 mentions)
Lc ms grade formic acid, by Thermo Fisher Scientific (1 mentions)
Quercetin, by Merck Group (1 mentions)
Kaempferol, by Merck Group (1 mentions)
Rutin, by Merck Group (1 mentions)
Myricetin, by Merck Group (1 mentions)
7 protocols using mouse il 18 elisa kit
1
Asbestos-induced Cytokine Release
2
Cytokine Quantification by ELISA
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The concentration of IL-1β, IL-6, and TNF-α in the serum and cell culture were detected by mouse IL-1β, IL-6, and TNF-α ELISA Kit (Excell, China) according to the manufacturer’s instructions. The concentration of IL-18 was detected by the mouse IL-18 ELISA Kit (R&D, DY7625-05).
3
Colonic Cytokine and Nitric Oxide Profiling
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Serum was collected from blood drawn by cardiac puncture at the end of the treatment. Explants from the proximal and distal colons of treatment and control groups (n = 3) were cultured overnight in RPMI 1640. Culture supernatants were measured for Nitrite by the Griess reaction method (Sigma G4410) as an index of Nitric Oxide generation.
Cytokine concentrations in neat culture supernatants and serum were determined using mouse Bio-Plex mouse cytokine 23-plex panel kit (Bio-Rad #M60009RDPD) and analysed using Luminex 200 (Bio-Rad) and Bio-Plex Manager software (Bio-Rad). IL18 was determined by 5 times diluted supernatant measured by a mouse IL18 ELISA kit (7625, R&D Systems). The most significantly altered cytokines are presented as pg per g of tissue.
Cytokine concentrations in neat culture supernatants and serum were determined using mouse Bio-Plex mouse cytokine 23-plex panel kit (Bio-Rad #M60009RDPD) and analysed using Luminex 200 (Bio-Rad) and Bio-Plex Manager software (Bio-Rad). IL18 was determined by 5 times diluted supernatant measured by a mouse IL18 ELISA kit (7625, R&D Systems). The most significantly altered cytokines are presented as pg per g of tissue.
4
Quantitative Analysis of Flavonoids
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Cyclophosphamide was purchased from Baxter Oncology GmbH (Batch No. 5J078A, Halle, Germany). Guinea pig serum was purchased from Beijing Bersee Bio Co., Ltd. Mouse IL-18 ELISA kit was purchased from R&D Systems (Minneapolis, USA). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Lactate Dehydrogenase (LDH) Cytotoxicity Detection Kit was purchased from TaKaRa (Otsu, Japan). LC/MS grade formic acid and methanol were purchased from Fisher Scientific (Fair Lawn, NJ). HPLC grade water was prepared from distilled water using a Milli-Q system (Millipore Laboratory, Bedford, MA). Rutin, quercetin, tricetin, myricitrin, kaempferol, myricetin, apigenin, and luteolin reference standards were obtained from Sigma-Aldrich (St Louis, MO).
5
Proinflammatory Cytokine Quantification
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Levels of proinflammatory cytokines, IL-1β, IL-6, IL-18, and tumor necrosis factor alpha (TNFα), were determined in cell culture medium at 0 h, 8 h, and 24 h following asbestos exposure using enzyme-linked immunosorbent assays (ELISA) as previously described [17 (link)]. Samples were run undiluted in triplicate, and assays were performed according to manufacturer’s instructions. Levels of IL-1β, IL-6, IL-18, and TNFα are reported as picograms per milliliter (pg/mL) of culture medium. ELISA kits (TNFα and IL-1β) were purchased from BD biosciences (San Jose, CA, USA), MBL International (Woburn, MA, USA) (mouse IL-18 ELISA Kit), and R&D systems (Minneapolis, MN, USA) (mouse IL-6 Quantikine ELISA Kit).
6
Cytokine and Cell Viability Evaluation in Cultured BV2 Cells
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Cytokines, including interleukin-1β (IL-1β) and interleukin-18 (IL-18) in perihematomal brain tissues and cell supernatant (collected from cultured BV2 cells supernatant in an in vitro experiment at 48 hours after the cultured BV2 cells reached 80% confluency), were evaluated using enzyme linked immunosorbent assay (ELISA) kits. The kits that were used are as follows: mouse IL-1β ELISA kit (MLB00C, R&D Systems, Minneapolis, MN, USA), mouse IL-18 ELISA kit (DY7625, R&D Systems), rat IL-1β ELISA kit (RA20020, Bio SWAMP, Wuhan, China), and rat IL-18 ELISA kit (RA20058, Bio SWAMP).
The neuronal cell viability and death rates were estimated using a cell counting kit-8 (CCK8) kit (CK04, Dojindo, Tokyo, Japan) and lactate dehydrogenase (LDH) assay kit (KGT02448, KeyGen, Nanjing, China), as described previously (Hu et al., 2020). For CCK8 assays, 10 μL of CCK8 reaction solution was added to the co-cultured neuronal cells per well, and the cells were incubated at 37°C for 1 hour. Absorption in each well was estimated at 450 nm using a multifunction measuring instrument (Thermo Fisher Scientific). For LDH assays, each sample was incubated with the corresponding reaction solution in accordance with the manufacturer’s instructions, and the absorption of each well was estimated at 440 nm using a multifunction measuring instrument (Thermo Fisher Scientific).
The neuronal cell viability and death rates were estimated using a cell counting kit-8 (CCK8) kit (CK04, Dojindo, Tokyo, Japan) and lactate dehydrogenase (LDH) assay kit (KGT02448, KeyGen, Nanjing, China), as described previously (Hu et al., 2020). For CCK8 assays, 10 μL of CCK8 reaction solution was added to the co-cultured neuronal cells per well, and the cells were incubated at 37°C for 1 hour. Absorption in each well was estimated at 450 nm using a multifunction measuring instrument (Thermo Fisher Scientific). For LDH assays, each sample was incubated with the corresponding reaction solution in accordance with the manufacturer’s instructions, and the absorption of each well was estimated at 440 nm using a multifunction measuring instrument (Thermo Fisher Scientific).
7
Cytokine Measurement in Cell Supernatants
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In each experiment, the supernatant or serum from each subgroup of cells was collected to measure the levels of circulating IL-6, IL-1β, IL-18, and TNF-α. The evaluation was performed using commercial kits from R&D Systems (Minneapolis, USA), which included the mouse IL-1β ELISA kit, mouse IL-18 ELISA kit, mouse IL-6 Quantikine ELISA kit, and mouse TNF-α Quantikine ELISA kit.
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