To detect the cell proliferation, BrdU incorporation assay was carried out with the BrdU ELISA kit from Abcam (Cambridge, MA, USA) according to the manual instructions [22 (link)]. The methyl thiazolyl tetrazolium (MTT) assay was conducted to measure the cell viability. HCC cells were grown at a concentration of 104 per well in 96-well plates with 200 μl medium for 24, 48, 72 and 96 h. After removing culture medium, HCC cells were incubated with 0.25 mg/ml MTT in serum-free medium at 37°C for 4 h. The supernatant was removed and 200 μl of dimethyl sulfoxide was added to solubilize the formazan. The plates were shaken for 5 min and cell survival was assessed by measuring the value of OD570 with a colorimetric reader. The experiment with 6 replicates was conducted for three times independently.
Brdu elisa kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The BrdU ELISA kit is a quantitative immunoassay designed to measure the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of proliferating cells. The kit provides the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
Infinite m200 plate reader, by Tecan (1 mentions)
Opsys mr, by Dynex (1 mentions)
Nanoquant infinite m200 pro reader, by Tecan (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti phospho gsk3β ser9, by Cell Signaling Technology (1 mentions)
Complete his tag purification resin, by Roche (1 mentions)
Anti human trem2, by Fujifilm (1 mentions)
Tev protease, by Promega (1 mentions)
Anti total gsk 3β, by Cell Signaling Technology (1 mentions)
Akt inhibitor ly294002, by Merck Group (1 mentions)
Protein a agarose, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Spectrostar nano, by BMG Labtech (1 mentions)
11 protocols using brdu elisa kit
1
Cell Proliferation and Viability Assays
2
BrdU Proliferation Assay for PDGF-Stimulated Cells
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The BrdU assay was performed using the 5-bromodeoxyuridine (BrdU) ELISA Kit (ab126556, Abcam). Briefly, the cells were seeded in a 96-well plate at 5 × 103 cells per well and were stimulated with 20 ng/mL PDGF (Thermo Fisher Scientific) for 24 h under starvation conditions. The cells were incubated with BrdU reagent for 6 h before the end of PDGF stimulation. After incubation with fixing solution, the anti-BrdU monoclonal antibody was added for 60 min at room temperature, followed by incubation with peroxidase goat anti-mouse IgG conjugate. The cells were then incubated with tetraliethylbenzidine peroxidase substrate for 30 min. After terminating the reaction by adding the stop solution, the absorbance was determined at 450 nm using a Tecan Infinite m200 plate reader.
3
Proliferation Assay for Breast Cancer Cells
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Proliferation was assessed in the MCF-7 and MDA-MB-231 breast adenocarcinoma cell lines and in the primary (HMEpC) breast cells using a colorimetric BrdU ELISA kit (ab126556, Abcam, Cambridge, UK). Each assay was performed in triplicate. MCF-7 cells were plated at a density of 2 × 105 cells/ml in 96-well plates and left to attach for 24 hours. The cells were then treated with subtilisin and α-chymotrypsin for 2, 6 or 24 hours. BrdU reagent was added to the cells at 2, 6 and 24 hours to evaluate proliferation. A series of wells on each plate were left untreated with BrdU as a background reading. The cells were fixed and the DNA denatured before assaying. Briefly, cells were washed, incubated with detector antibody for 1 hour, washed again and then incubated with peroxidase conjugate secondary antibody for 1 hour. Following a final wash step, cells were incubated for 30 mins with TMB peroxidase substrate. Stop solution was added to the wells and plates read in a microplate reader (Opsys MR, Dynex Technologies, Worthing, UK) at dual wavelengths of 450/550 nm. Proliferation was expressed as the mean optical density of BrdU positively labelled cells/BrdU negative cells.
4
Investigating HUVEC Proliferation: TGF-β and IL-37
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For the combined effect of TGF-β and IL-37 on HUVECs proliferation, 1 × 104 HUVECs in 100 μl of ECM were seeded in 96 well plates at 1 × 104/ml in serum-free ECM containing 0.1% BSA for overnight. The cells were then stimulated with combinations of different concentrations of IL-37 (0, 0.1, and 1 ng/ml) and TGF-β (0, 0.1, 1 and 10 ng/ml) for 48 hours. For the bioactivity of biotinylated IL-37, 1 × 104 HUVECs in 100 μl of ECM were seeded in 96-well plates and maintained ECM containing 5% FBS. The cells were stimulated with different concentrations of biotinylated or unbiotinylated IL-37 or (0, 0.1, 0.5 1 and 5 ng/ml). For the effect of IL-37 on HUVECs proliferation, 1 × 104 HUVECs in 100 μl of ECM were seeded in 96-well plates and maintained ECM containing 5% FBS without supplemented growth factors unless otherwise indicated. Antibodies neutralizing TGF-β (R&D Systems, Minneapolis, MN) or ALK1 (R&D Systems, Minneapolis, MN) were used at 10 μg/ml to block the effect of IL-37. Cell proliferation was determined by BrdU ELISA kit (Abcam, Cambridge, MA) according to company’s instructions. The statistical significance was determined by Student’s t-test.
5
Quantifying Cell Viability and Proliferation
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The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to measure cell viability. For the MTT assay, HCC cells were grown at concentrations of 5 × 104 cells per well in 96-well plates overnight. After removal of the culture medium, 150 μl dimethyl sulfoxide was added to each well. The 96-well plate was shaken for 5 min and optical density was measured at a wavelength of 570 nm using a microplate colorimetric reader. The BrdU incorporation assay was performed to measure cell proliferation using the BrdU ELISA kit from Abcam (MA, USA).
6
Cell Proliferation Assay with BrdU
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Cell proliferation was assessed with a colorimetric immunoassay based on the measurement of BrdU incorporation during DNA synthesis. A375P or A375DR cells were seeded in 96-well plates (2.9 × 103 cells/well). After 24 h, cells were incubated with or without 0.5 or 1 µM ONC for 24, 48, or 72 h, and, subsequently, for another 4 h with BrdU (BrdU Elisa Kit; Abcam, Milan, Italy). The medium was aspirated and cells were incubated with a fixing solution for 30 min. Plates were washed three times and the anti-BrdU detector antibody was added for 1 h. Plates were then washed a further three times, incubated for 30 min at room temperature with peroxidase goat anti-mouse IgG conjugate and then washed three times again. The tetramethybenzidine chromogenic peroxidase substrate was added, and plates were incubated for 30 min in the dark. Finally, a stop solution was added and the OD450 measured in a plate placed in the Tecan NanoQuant Infinite M200 Pro reader (Tecan Group Ltd., Männedorf, Switzerland).
7
Inhibition of Inflammatory Pathways
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p38-MAPK inhibitor (SB203580), ERK1/2 inhibitor (U0126), JNK inhibitor (SP600125), NF-κB inhibitor (Bay 11–7082), Akt inhibitor (LY-294002), and DAPI were purchased from Sigma-Aldrich. GM-CSF was purchased from R&D Systems. Kits for Cell Proliferation Assay (MTS), DeadEnd Fluorometric TUNEL System, and TEV protease were purchased from Promega. In Situ Cell Death Detection kit and cOmplete His-Tag Purification Resin were purchased from Roche. BrdU ELISA kit was purchased from Abcam. Protein A Agarose and Alexa Fluor 488 Phalloidin were purchased from Thermo Fisher Scientific. Antibodies for Western blotting used in this study, including anti–Phospho-NF-κB p65 (Ser536), anti–total-NF-κB p65, anti–Phospho-Akt (Ser473), anti–total-Akt, anti-Phospho-GSK3β (Ser9), anti–total-GSK3β, anti–β-catenin, and anti–β-actin, were purchased from Cell Signaling Technology; anti–human TREM2 antibody for Western blotting was purchased from R&D Systems (AF1828). Antibodies for immunofluorescence including anti-Iba1 and anti–human TREM2 were purchased from Wako and Santa Cruz Biotechnology (sc-373828), respectively.
8
BrdU ELISA Assay for MCF-7 Cell Proliferation
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Proliferation was assessed in the MCF-7 cells using a colorimetric BrdU ELISA kit (ab126556, Abcam, Cambridge, UK). Each assay was performed in triplicate. MCF-7 cells were plated at 2x105 cells/ml in 96-well plates and left to attach for 24 h before treatment with subtilisin (30, 100 and 300nM) or α-chymotrypsin (300 and 1000nM) for 24 h. BrdU reagent was added for the final 2, 6 and 24 h of incubation after which the cells were fixed and the DNA denatured. Briefly, cells were washed, incubated with detector antibody (1 h), washed again and incubated with peroxidase conjugated secondary antibody (1 h). After washing, cells were incubated with TMB peroxidase substrate and a stop solution was added before reading in a microplate reader (using a dual wavelength of 450/550 nm). Proliferation was expressed as the mean optical density of BrdU positively labelled cells/BrdU negative cells.
9
Microglial Proliferation Assay with sTREM2
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WT and Trem2-KO primary microglial cells were seeded into 96-well culture plates at a density of 6 × 104 cells/well and were cultured for 48 h after GM-CSF withdrawal. After administration with purified Fc or sTREM2-Fc fusion protein for 24 h, the cell proliferation was assessed using BrdU ELISA kit (Abcam) according to the manufacturer’s instruction.
10
Quantifying Caco-2 Proliferation with BrdU ELISA
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The proliferation of Caco-2 in cultures with SPF was quantified using a bromodeoxyuridine (BrdU) ELISA Kit (Abcam, Cambridge, UK). For this purpose cultures were maintained in a 96-well plate. The analysis were performed after 24 and 72 h of cell culture. Testing was performed according to the manufacturer’s instructions. The reaction was read using a spectrophotometer microplate reader (Spectrostar Nano, BMG Labtech, Ortenberg, Germany) at a wavelength of 450/550 nm.
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