VSMC proliferation was determined by either CCK-8 (Dojindo Molecular Technologies) assays or BrdU incorporation assays (Roche) according to the protocols provided by manufacturers. For the CCK-8 assay, 10 μl of CCK-8 was dispensed into each well of a 96-well culture plate, and the absorbance was measured at 450 nm after 2 h incubation. For the BrdU incorporation assay, BrdU was added to the culture medium for incorporation into cellular DNA. After 4 h of incubation, cells were fixed, and anti-BrdU antibody was added and incubated for 30 min followed by measurement of the absorbance at 450 nm.
Brdu incorporation assay
Manufactured by Roche
Sourced in Germany, Switzerland, United Kingdom, United States
The BrdU incorporation assay is a laboratory technique used to measure cell proliferation. It involves the incorporation of the synthetic thymidine analog bromodeoxyuridine (BrdU) into the DNA of dividing cells. The assay can be used to quantify the number of cells undergoing DNA synthesis, which is an indicator of cell proliferation.
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Lab products found in correlation
Microplate reader, by Bio-Rad (2 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
Mcdb 131 medium, by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin (pha), by Roche (1 mentions)
Wst 1 assay, by Roche (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Mts assay, by Promega (1 mentions)
Gallios flow cytometer system, by Beckman Coulter (1 mentions)
Kaluza analysis software, by Beckman Coulter (1 mentions)
Pd98059, by Merck Group (1 mentions)
96 well microtiter plate, by Corning (1 mentions)
Mtt assay, by Bio-Rad (1 mentions)
Mtt assay, by Bio-Techne (1 mentions)
Lysis buffer, by Solarbio (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
Mab3222, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Ascorbic acid, by Thermo Fisher Scientific (1 mentions)
Infinite 200 luminometer, by Tecan (1 mentions)
38 protocols using brdu incorporation assay
1
VSMC Proliferation Assays
2
Measuring Cell Proliferation and Migration
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Cell proliferation was measured using a bromodeoxyuridine (BrdU) incorporation assays (Roche Diagnostics, Burgess Hill, UK), performed in a 96-well plate with 2000 cells/well as previously described [18] (link), [27] (link). For BrdU assays, cells were treated with inhibitors for 16 h prior to assay. Scratch wound healing assays were performed as previously described [18] (link). Briefly, HUVECs were grown to confluence and starved for 3 h in serum-free MCDB131 medium (Invitrogen, Amsterdam, Netherlands) containing 0.2% (w/v) BSA. Cells were pre-treated with JK-31 for 1 h prior to making a vertical scratch wound through the cell monolayer with a sterile 200 µl plastic pipette tip of ∼0.9 mm tip width. Wounded cell monolayers were washed once with PBS and photographed. Cells were stimulated with 25 ng/ml VEGF-A for 16 h in the presence or absence of JK-31 and wounded cell monolayers photographed once more. Wound widths were measured using Image J software and % wound closure calculated by ((width before – width after)/width before) ×100.
3
Lymphocyte Proliferation Assay with Fractions
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The effect of various concentrations of the fractions on proliferation of phyto-hemagglutinin (PHA)-activated lymphocytes was determined by BrdU incorporation assay (Roche, Germany). Ten mL heparinized blood sample was collected from 25-30 year healthy volunteers (with their consent) and peripheral blood lymphocytes (PBLs) were isolated using Ficoll-hypaque (Bahar-afshan, Iran) gradient centrifugation. After washing, lymphocytes were seeded in 96-well plates (1×105 cells/well). Then, 10 μL PHA (Fluka, Germany) (1/80) and various concentrations of the fractions (0.01-200 μg/mL) were added in a final volume of 100 μL. Negative control was PHA-stimulated cells treated with DMSO as the solvent at the highest concentration used in the test wells (e.g., 0.1%) (PHA-only treated cells). After addition of BrdU for 18 h, DNA was denatured and the cells were incubated with anti-BrdU monoclonal antibody. Then the optimal density (OD) was measured with an enzyme-linked immunosorbent (ELISA) microplate reader (Biotek Inc., USA) at 450 nm. The experiments were plated in triplicate wells and performed at least three times. The percentage of proliferation was determined as follows:
Percentage of proliferation = (OD of treated cells/OD of negative control) × 100
Percentage of proliferation = (OD of treated cells/OD of negative control) × 100
4
KFB proliferation response to FAs
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KFBs were seeded into 96-well flat bottom plates and cultured in culture medium
with 10% FBS. After 24 h, FAs were added and the cells were incubated for 48 h,
followed by analysis using BrdU incorporation assay (Roche, Basel, Switzerland),
according to the manufacturer's instructions.
with 10% FBS. After 24 h, FAs were added and the cells were incubated for 48 h,
followed by analysis using BrdU incorporation assay (Roche, Basel, Switzerland),
according to the manufacturer's instructions.
5
PGE2 Proliferation Assay in RL95-2 Cells
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RL95-2 Cells were seeded in 96-well plates at 5 × 103 cells/well in sextuplicate. The next morning cells were incubated with different concentration of PGE2 or sulprostone for 48 h. 5-Bromo-2'-Deoxyuridine (BrdU) incorporation assay (11647229001, Roche) was used to determine the cell proliferation according to manufacturer’s protocols. OD was quantified at 450 nm using Elx800 universal Microplate Reader.
6
BrdU Proliferation Assay in HCC1937
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HCC1937 cells were seeded on 96 well-plate and transfected to measure the cell proliferation rate. The 5-Bromo-2’-Deoxyuridine (BrdU) incorporation assay (Roche, Basel, Switzerland) was used according to the manufacturer’s protocol. The optical density was measured at 450 nm using Elx800 universal Microplate Reader. Each experiment was repeated three times (n = 3).
7
Cell Proliferation Assays for Myoblasts
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Two different assays were used to assess cell proliferation according to the manufacturer's instructions: a water-soluble tetrazolium 1 (WST-1) assay and a bromodeoxyuridine (BrdU) incorporation assay (both from Roche Molecular Biochemicals, Mannheim, Germany). Myoblasts were seeded at 5000 cells per well in a 96-well plate. Cells were either treated for five consecutive days with G-CSF or for 24/48 h with G-CSF and palmitate alone or in combination prior to the assay.
8
BrdU Cell Proliferation Assay
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Cell proliferation was assessed using the BrdU incorporation assay (Roche, Mannheim, Germany). Briefly, cells were seeded into 96-well plates at an initial density of 4 × 103 cells/well. BrdU labeling solution (10 μL/well) was added to the cells at the indicated time points. After a 2 h incubation, the culture medium was removed and the cells fixed. The DNA was denatured with FixDenat (200 μL/well) and anti-BrdU-POD working solution (100 μL/well) was then added to the cells and incubated for 90 min. Immune complexes were detected by the subsequent substrate reaction. The reaction product was quantified by measuring the absorbance at 370 nm (reference wavelength: approximately 492 nm).
9
Glioma Cell Proliferation Assay
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Proliferation of glioma U-87 MG and LN18 cells was measured in cells cultured in 96-well plates (seeded at the density of 3 × 103 per well) using BrdU incorporation assay (Roche). LN18 or U87 cells were cultured in the presence of HDACi for 24 or 48 h after gene silencing. Then, BrdU was added for 2 h and BrdU incorporation was measured according to the vendor’s protocol.
10
Cell Proliferation Assay with RMECs
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RMECs were seeded at a density of 1 3 10 4 cells/well on 1% gelatin-coated 96-well plates and cultured for 24 hours in DMEM containing 10% PS. The media then was refreshed with media containing test compounds and the cells incubated for a further 24 hours. Cell proliferation was determined using a bromodeoxyuridine (BrdU) incorporation assay (Roche, Mannheim, Germany) as described previously. 12
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