Cell proliferation was measured by BrdU incorporation, using a cell proliferation ELISA kit (Roche). Cells were plated at 2500 cells per well in opaque 96-well flat-bottom plates and drugs were added with fresh media 24 hours after plating. Cells were incubated in drug for 48 hours, with BrdU added to the media for the last 24 hours. Luminescence was detected using a Veritas microplate luminometer (Turner Biosystems). The drugs were imatinib, sunitinib, RAD001 (everolimus) and GDC-0941 (all from LC laboratories); and PD-0332991 (palbociclib) and LEE011 (from ChemieTek). All drugs were dissolved in DMSO. DMSO controls were incorporated in all studies, as solvent-only comparators.
Cell proliferation elisa kit
Manufactured by Roche
Sourced in Germany, Switzerland, United States, France, United Kingdom
The Cell Proliferation ELISA kit is a laboratory tool used to quantitatively measure cell proliferation in vitro. It is based on the measurement of bromodeoxyuridine (BrdU) incorporation during DNA synthesis.
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Lab products found in correlation
Fixdenat solution, by Roche (5 mentions)
Peroxidase conjugated mouse anti brdu monoclonal antibody, by Roche (5 mentions)
Veritas microplate luminometer, by Promega (2 mentions)
Microplate reader, by Molecular Devices (2 mentions)
Microplate reader, by Agilent Technologies (2 mentions)
Centro lb 960 luminometer, by Berthold Technologies (2 mentions)
Rad001, by LC Laboratories (1 mentions)
Pd 0332991, by Chemietek (1 mentions)
Imatinib, by LC Laboratories (1 mentions)
Gdc 0941, by LC Laboratories (1 mentions)
Sunitinib, by LC Laboratories (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Hkbml, by RIKEN BioResource Center (1 mentions)
Thiazolyl blue tetrazolium bromide mtt, by Merck Group (1 mentions)
Nitrocellulose membrane, by Santa Cruz Biotechnology (1 mentions)
Honokiol, by Nacalai Tesque (1 mentions)
Cdc2 p34, by Santa Cruz Biotechnology (1 mentions)
Leupeptin, by Roche (1 mentions)
Pepstatin, by Roche (1 mentions)
Pdgf bb, by ProSpec (1 mentions)
82 protocols using cell proliferation elisa kit
1
Cell Proliferation Assay with Drugs
2
HUVEC Proliferation Assay and Cell Cycle Analysis
3
HKBML Cell Proliferation Assay
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The human brain-derived lymphoma cell line, HKBML (Riken BioResource Center, Tsukuba, Japan), was maintained in Ham's nutrient mixture F-12 medium (Sigma-Aldrich; KGaA, Darmstadt, Germany; cat. no. N6658) supplemented with 15% fetal bovine serum and penicillin-streptomycin, and incubated in a 37°C, 5% CO2 and a humidified atmosphere. Prior to stimulation with 0, 250, 500 or 1,000 ng/ml MCP-1, the cells were preincubated in F-12 medium containing 0.5% fetal bovine serum overnight. HKBML proliferation activity was measured by 5-bromo-2′-deoxyuridine (BrdU) incorporation into the cells, which were plated as floating single cells at 2×104 cells per well onto 96 well plates. Following a 3 day incubation with MCP-1, BrdU (10 µM) was added for 2 h at 37°C and in a 5% CO2 humidified atmosphere, using the Cell Proliferation ELISA kit (Roche Diagnostics, Basel, Switzerland; cat. no. 11647229001), according to the manufacturer's instruction. The media and no cells was used as a negative control.
4
Honokiol Inhibits Apoptosis and Cell Cycle
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Honokiol (98% purity) was purchased from Nacalai Tesque (Kyoto, Japan). Thiazolyl blue tetrazolium bromide (MTT) and other chemicals of analytical grade were purchased from Sigma Chemical Co. (St. Louis, MO). Leupeptin, pepstatin, and cell proliferation ELISA kit were purchased from Roche Diagnostics GmbH (Mannheim, Germany). Primary antibodies for procaspase 3, caspase 8, caspase 9, CDK6, p53, and cleaved PARP were purchased from Cell Signaling Technology (Beverly, MA). Primary antibodies for cyclin D1, cyclin D2, cyclin E, CDK-2, CDK-4, cyclin A, Cdc2p34, PCNA, caspase 3, anti-mouse IgG horseradish peroxidase-linked and anti-rabbit IgG horseradish peroxidase-linked secondary antibodies, and nitrocellulose membranes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Kip1/p27 antibody was purchased from BD-Pharmingen (San Diego, CA), cyclin B1 and anti-Cip1/p21 antibody from Upstate Biotechnology (Lake Placid, NY).
5
PDGF-BB Stimulated SMC Proliferation Assay
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SMC DNA synthesis was evaluated based on the level of (BrdU incorporated. Primary mouse SMCs (5 × 103 per well) were growth-arrested in a 96-well microplate. After growing to 60% confluence, the cells were serum-starved for 24 h and subsequently treated with 20 ng ml−1 of PDGF-BB (ProSpec; Rehovot, Israel) for 48 h. BrdU was added for the last 2 h of treatment. BrdU incorporation was determined using a cell proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany) in accordance with the manufacturer’s protocol.
6
RASMC Proliferation Assay with Pemafibrate and Bezafibrate
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For evaluation of the cell proliferation of RASMCs, a BrdU incorporation assay was demonstrated using a Cell Proliferation ELISA kit (1647229; Roche Applied Science, Mannheim, Germany). RASMCs were cultured at 4000 cells/well in 96-well culture plates in complete media (n = 5). At 60%–70% confluence, cells were maintained in serum-free media for 24 h. At 12 h before serum stimulation, cells were treated with 0–1000 nM pemafibrate, or 0–1000 μM bezafibrate and subsequently stimulated with 10% FBS for 48 h. A BrdU (10 μM) solution was added during the last 2 h of incubation. Subsequently, the cells were dried and fixed, and cellular DNA was denatured with FixDenat solution (Roche Applied Science) for 30 min at room temperature. A peroxidase-conjugated mouse anti-BrdU monoclonal antibody (Roche Applied Science) was added to the culture plates, followed by incubation for 90 min at room temperature. Finally, a tetramethylbenzidine substrate was applied for 15 min at room temperature, and the absorbance of samples was measured using a microplate reader at 450–620 nm. Mean data were expressed as a ratio of the control (untreated) cell proliferation.
7
Cell Proliferation Assay using BrdU
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We used 5-bromo-2-deoxyuridine (BrdU) assay to evaluate cell proliferation. After receiving indicated treatment for cells, 10 mM BrdU was added and followed by incubation for 2 h. The nuclear incorporation of BrdU was measured using a cell proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany).
8
Anti-Proliferative Effect of FFE Evaluated by ELISA
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A cell proliferation ELISA kit (Roche, Basel, Switzerland) was used to evaluate the anti-proliferative effect of FFE treatment according to the manufacturer’s instructions. After 24-, 48-, and 72-h treatment with FFE, bromodeoxyuridine (BrdU, 10 μL/well) was added to each well and incubated for 4 h at 37 °C. The BrdU solution was removed, and 200 μL of FixDenat was added to each well and incubated for 30 min. The reacted FixDenat solution was removed, and 100 μL of anti-BrdU-peroxiase (POD) was added to each well. After washing with PBS three times, 100 μL of substrate solution was added to each well, and the optical density was measured at 450 nm using a microplate reader (Molecular Devices).
9
BrdU Incorporation Assay for Cell Proliferation
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The BrdU incorporation assay was performed as previously described (22 (link)). In brief, 5,000 cells per well were seeded in 96-well microplates and allowed to attach overnight. The incorporation of BrdU into newly synthesized DNA was then examined with a cell proliferation ELISA kit (Roche), according to the manufacturer's instructions. A microplate reader was used to determine the optical density of each well at 450/595 nm.
10
Quantifying Cell Proliferation and Viability
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For quantification of cell proliferation, cells in 96-well plates were cultured overnight with medium containing 5-bromo-2’-deoxuridine (BrdU). BrdU incorporation was determined colorimetrically with the Cell proliferation ELISA kit (Roche, Basilea, Switzerland) following the manufacturer’s instructions. For assessing cell viability, cells grown in 96-well plates were incubated with medium containing 0.75 mg/ml of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 3 h at 37 °C. The resulting formazan crystals were solubilized in Isopropanol/0.04 N HCl solution and optical density was read at 575 and 650 nm using a Synergy HT reader (BIO-TEK Instruments, Winooski, VT, USA). The OD (575-650) was expressed relative to control fibroblasts, which were given the value of 100%.
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