To analyse differences in cellular proliferation, when exposed to the environmental factors in the FFED, the BrdU assay (Abcam) was performed at 24 h and 120 h (5 days) post induction of differentiation. 24 h prior to the read-out, 1X-BrdU reagent was added to the cell culture vessels for incorporation by incubation at 37 °C and 5% CO2. For the read-out, the culture media was aspirated, and cells were fixed with the supplied fixing solution. This was followed by exposure to the anti-BrdU antibody (primary antibody), followed by incubation at room temperature for 1 h and washing with the supplied plate wash buffer. The cells were incubated with the HRP-tagged secondary antibody at room temperature for 30 min followed by TMB exposure and recording absorbance at 450 nm. Quantification of percentage proliferation was done in R (v4.1.2)58 .
Brdu assay
Manufactured by Abcam
Sourced in United Kingdom, United States
The BrdU assay is a laboratory technique used to measure cellular proliferation. It detects the incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into the DNA of dividing cells, providing a quantitative assessment of DNA synthesis.
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Lab products found in correlation
Flexstation 3, by Molecular Devices (3 mentions)
Softmax pro, by Molecular Devices (3 mentions)
Matrigel chamber, by BD (1 mentions)
Mts assay, by Merck Group (1 mentions)
Elx808iu microplate reader, by Agilent Technologies (1 mentions)
Mtt assay, by Merck Group (1 mentions)
Aminocaproic acid, by Merck Group (1 mentions)
Mts assay, by Promega (1 mentions)
Prism 8, by GraphPad (1 mentions)
Fluoxetine, by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
8 protocols using brdu assay
1
Quantifying Cellular Proliferation in FFED
2
BrdU Assay for Stx2a Cytotoxicity
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BOECs were seeded in a 12-well plate at a density of 70,000 cells per well. After 24 h, Stx2a in a concentration of 0.1–10 ng/mL together with 10 µM BrdU were added and stored for 24 h in a 37 °C incubator with 5% CO2. Next, the BrdU assay was performed according to the manufacturer’s protocol (Abcam, Cambridge, UK).
3
Cell Proliferation, Migration, and Invasion Assays
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A bromodeoxyuridine (BrdU) assay (Abcam, USA) was used to test cell proliferation ability following the manufacturer's protocols. Briefly, 769‐P and ACHN cells were seeded into 6‐well plates under BrdU‐labelling solution for 24 h at 37°C in a CO2 incubator. Then, the cells were fixed and permeabilized according to the standard immunocytochemistry and photographed under a microscope. A wound healing assay was used to test the capacity of cell migration. Briefly, 769‐P and ACHN cells were seeded into six‐well plates and incubated with serum‐free medium. A 200‐μl plastic pipette tip was utilized to make a scratch after the density of the cells reached 80%, and the migration distance was photographed after 24 h. A Matrigel chamber (BD Biosciences, USA) was used to detect cell invasion ability according to the manufacturer's protocols. Briefly, cells were seeded into the upper chamber in serum‐free medium, and the lower chamber was filled with 10% fetal bovine serum. After 24 h, the non‐migrated cells on the upper side of the chamber were removed and stained with crystal violet. Finally, the cells were photographed and analysed under a microscope.
4
Cytotoxicity and Proliferation Assays of PAC in Cells
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Cells were incubated in 96-well plates and exposed to the indicated concentration of PAC (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 μM) or vehicle control (DMSO) for 24 h. Cell viabilities were evaluated with an MTT assay (Sigma, USA). In brief, 10 μL of 5 mg/mL MTT was added to each well and incubated for 4 h. The medium was replaced with 150 μL of DMSO to dissolve the crystal formazan dye and absorbance was detected at 540 nm using an ELX808IU Microplate Reader (BioTek, USA).
Cells were seeded in 96-well plates and exposed to the indicated concentration of PAC (0.1, 0.2, 0.3, 0.4, 0.6 μM) or vehicle control (DMSO) for 24 h. Cell proliferation was measured with a BrdU assay (Abcam UK): BrdU (10 μM) was added to each well and incubated for 12 h, and the BrdU signal was calculated after absorbance detection at 450 nm.
Cells were plated in 96-well plates and exposed to the indicated concentrations of PAC (0.2, 0.4, 0.6 μM) or vehicle control (DMSO) for 12, 24, and 36 h. Cell viability was examined with an MTS assay (Sigma, USA), 20 μL of MTS was added to each well and incubated for 1 h, after which the absorbance of the MTS signal was calculated after absorbance detection at 490 nm.
Cells were seeded in 96-well plates and exposed to the indicated concentration of PAC (0.1, 0.2, 0.3, 0.4, 0.6 μM) or vehicle control (DMSO) for 24 h. Cell proliferation was measured with a BrdU assay (Abcam UK): BrdU (10 μM) was added to each well and incubated for 12 h, and the BrdU signal was calculated after absorbance detection at 450 nm.
Cells were plated in 96-well plates and exposed to the indicated concentrations of PAC (0.2, 0.4, 0.6 μM) or vehicle control (DMSO) for 12, 24, and 36 h. Cell viability was examined with an MTS assay (Sigma, USA), 20 μL of MTS was added to each well and incubated for 1 h, after which the absorbance of the MTS signal was calculated after absorbance detection at 490 nm.
5
Cell Proliferation Assay with Aminocaproic Acid
6
Anti-CEMIP shRNA Modulates FLS Viability
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FLS were treated with anti-CEMIP shRNA or control shRNA and stimulated or not with TGF-β for 2 days. Cell viability was assessed by using MTS assay (Promega, Madison, Wisconsin, USA) and cell proliferation was measured by using BrdU assay (Abcam) according to the manufacturer’s instructions. Results are expressed as a percentage of surviving (for MTS test) or proliferating (for BrdU test) cells compared with control cells.
7
BrdU Proliferation Assay Protocol
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Cells were seeded in 96-well plates at a density of 2 × 104 cells per well. BrdU assay (Abcam, Cambridge, UK) was performed according to the manufacturer’s instruction. Wells were read on a Flex Station 3 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm. Data were collected with Soft Max Pro (Molecular Devices) software. Means and standard error mean (SEM) were calculated in GraphPad Prism 8 software (GraphPad Software, Inc., La Jolla, CA, USA).
8
BrdU Assay for Cell Proliferation
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