HRMECs were seeded at a density of 1 × 104 cells/well in 96-well plates and cultured for 24 hours in CSC complete media. Media was then replaced with fresh CSC media containing test substances, and the cells were incubated for a further 24 hours. BrdU was then added and its incorporation into HRMEC DNA measured using a colorimetric cell proliferation ELISA assay (Roche Diagnostics).
Colorimetric cell proliferation elisa assay
Manufactured by Roche
Sourced in Germany
The Colorimetric cell proliferation ELISA assay is a laboratory equipment product designed to measure cell proliferation. It utilizes a colorimetric detection method to quantify the amount of a specific substance, such as a protein, in a sample. The core function of this assay is to provide a means for researchers to assess cell growth and proliferation in a variety of experimental settings.
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5 protocols using colorimetric cell proliferation elisa assay
1
HRMEC Proliferation Assay Protocol
2
Cell Proliferation Assay with CLO Extract
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The BrdU incorporation was used to analyse cell proliferation activity monitored by quantification of BrdU introduced to the genomic DNA during cell growth after 72‐hrs CLO extract (c = 50–1000 μg/ml) treatment. DNA synthesis was assessed using colorimetric cell proliferation ELISA assay (Roche Diagnostics GmbH, Mannheim, Germany) following manufacture protocol. The colour intensity was measured with Cytation™ 3 Cell Imaging Multi‐Mode Reader (Biotek) at 450 nm (reference wavelength: 690 nm). The results were expressed as a percentage of the control. All experiments were performed in triplicate including untreated controls and vehicle (ethanol) internal controls. For following analyses, final concentration 450 μg/ml was used.
3
BrdU Proliferation Assay for Wt1KO Cells
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EC proliferation was quantified using a BrdU incorporated colorimetric cell proliferation ELISA assay (Roche Diagnostics). Briefly, 5000 control and Wt1KO cells were plated in gelatin-coated 96-well culture plates, with four replicas per condition. After 8 h of seeding, cells were synchronized in a serum-free EBM-2 medium. Cells were then incubated with BrdU labelling solution for another 24 h at 37°C in complete EBM-2 medium followed by fixation and incubation with anti-BrdU peroxidase conjugate for an additional 1.5 h at room temperature. Finally, after substrate reaction, colour intensity was measured with an Infinite 200 plate reader at 450 nm (reference wavelength: 690 nm).
4
Cell Proliferation Assays for EOT Treatment
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BrdU incorporation was used to analyze cell proliferation activity and was monitored by quantification of BrdU introduced to the genomic DNA during cell growth after EOT treatment (final dilutions ranged between 0.72–0.023 µg/mL and 0.72–0.0056 µg/mL, respectively). DNA synthesis was assessed using a colorimetric cell proliferation ELISA assay (Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer’s protocol. The color intensity was measured with a CytationTM 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA at 450 nm (reference wavelength: 690 nm). The results were expressed as a fold of the control. All experiments were performed in triplicate. For the following analyses final dilutions (calculated from MTS and BrdU assays) of 0.12 µg/mL (MDA-MB-231 cells) or 0.13 µg/mL (MCF-7 cells) were used.
5
Cell Proliferation Analysis via BrdU Incorporation
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The BrdU incorporation was used to analyze cell proliferation activity monitored by quantification of BrdU introduced to the genomic DNA during cell growth after EOC treatment (final dilution in range 1:1250–1:40,000 resp. – 1:160,000). DNA synthesis was assessed using colorimetric cell proliferation ELISA assay (Roche Diagnostics GmbH, Mannheim, Germany) following manufacture protocol. The color intensity was measured with CytationTM 3 Cell Imaging Multi-Mode Reader (Biotek) at 450 nm (reference wavelength: 690 nm). The results were expressed as a fold of the control. All experiments were performed in triplicate. For following analyses, final dilutions (calculated from MTS and BrdU assays) of 1:65000 (MDA-MB-231 cells) or 1:25000 (MCF-7 cells) were used.
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