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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Published: September 3rd, 2016

DOI:

10.3791/54350

1Medical Genetics, Hunter Medical Research Institute, 2Priority Research Centre for Cancer, School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, 3Pathology North, John Hunter Hospital

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

The tumor suppressor gene, p53, is an essential regulator of a number of cellular processes, including cell cycle arrest, apoptosis and senescence1. It is responsible for maintaining genomic stability, and is therefore crucial for maintaining the balance of cell death and cell growth. Mutations in p53 are common in cancer and are the major cause of p53 inactivation leading to uncontrolled cancer cell proliferation2. Interestingly, mutations in p53 only account for approximately 25% of breast cancers3, suggesting that other mechanisms are responsible for the loss of p53 function. The recently discovered p53 isoforms have been shown to b....

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1. Preparing Cells for Proliferation Assays

Note: Prepare the two cell lines in the same manner and seed in the same format for each method to be analyzed.

  1. Grow MCF-7-LeGO and MCF-7-Δ40p53 7 cells to 75-80% confluence in T 75 cm2 tissue culture flasks using phenol-red free Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine, 2 µg/ml insulin and 1 µg/ml Puromycin at 37 °C with 5% CO<.......

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To study different methods of measuring the proliferation of cultured cells, the cell proliferation of MCF-7-Δ40p53 transduced cells was compared to the non-transduced MCF-7-LeGO breast cancer cell line. The three methods that were compared – the conventional hemocytometer method, cell viability luminescence assay, and cell imaging analysis- are outlined in the schematic diagram (Figure 1). Each method has advantages and disadvantages to accurately measure.......

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In this protocol three different methods of measuring cell proliferation in cultured cells were examined. Each method was capable of reproducible and accurate measurements of cell proliferation over 96 hr, and the results were comparable between each of the methods tested (Figure 2 and 3). Both the luminescence-based assay and cell imaging method produced the most robust results, showing linear increases in cell proliferation after 96 hr (Figure 2b, c). Additionally, cel.......

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We would like to thank Dr Hamish Campbell and Prof Antony Braithwaite for their help in developing the transduced MCF-7-LeGO cell lines. We would like to acknowledge our funding support by the Bloomfield Group Foundation through the Hunter Medical Research Institute. B.C.M is supported by an APA scholarship through the University of Newcastle and the MM Sawyer Scholarship through the Hunter Medical Research Institute.

....

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Name Company Catalog Number Comments
Dulbecco's Modified Eagle Medium, no phenol-red ThermoFisher Scientific 21063-045 Supplemented with 10% FBS, 200mM L-glutamine, 2µg/ml insulin and 1µg/ml puromycin
L-glutamine solution (100x) ThermoFisher Scientific 25030-081
Insulin solution human Sigma-Aldrich I9278-5ML
Fetal bovine serum (FBS) Bovogen Biologicals SFBS-F-500ml
Puromycin dihydrochloride Sigma-Aldrich P9620-10ML
0.5% trypsin-EDTA solution (10x) ThermoFisher Scientific 15400-054 Dilute to 2x in DPBS
Dulbecco's Phosphate Buffered Saline (DPBS) (1x) ThermoFisher Scientific 30028-02
Tissue culture flask, 75cm2 growth area Greiner Bio-One 658175
Scepter 2.0 Cell Counter Merck Millipore Automated cell counter
96 well multiwell plate, flat bottom Nunc 167008
Improved Neubauer Hemocytometer BOECO Germany BOE 01
Olympus IX51 inverted microscope Olympus IX51
CellTiter-Glo 2.0 Assay Promega G9242 Luminescence-based assay
Cytation 3 Cell Imaging Multi-Mode Reader BioTek Plate reader for luminescence, fluorescence and brightfield cell imaging
Gen5 Data Analysis Software BioTek GEN5

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