Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

Maciej Wakula; Anna Balcerak; Urszula Smietanka; Mateusz Chmielarczyk; Ryszard Konopi&#324;ski; Ewa  A. Grzybowska

doi:10.3791/61461

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

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Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

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06:46 min

June 24, 2020

DOI: 10.3791/61461-v

Maciej Wakula¹, Anna Balcerak¹, Urszula Smietanka¹, Mateusz Chmielarczyk¹, Ryszard Konopiński¹, Ewa A. Grzybowska¹

¹Maria Sklodowska-Curie National Research Institute of Oncology

Overview

This article details a protocol for quantifying focal adhesions and cell shape index in confluent monolayers of MCF7 cells using confocal imaging. The study highlights how variations in both adhesion and cell shape can be analyzed through accessible imaging techniques.

Key Study Components

Research Area

Cell Biology
Focal Adhesion Analysis
Cell Shape Quantification

Background

Focal adhesions are critical for cell adhesion and signaling.
Understanding cell shape and adhesion can impact the study of cancer biology.
MCF7 cells serve as a model system for breast cancer research.

Methods Used

Confocal microscopy for imaging cellular structures.
ImageJ software for analyzing focal adhesion areas and cell shape index.
Manual and automated analysis protocols for efficient data collection.

Main Results

Higher numbers and larger sizes of focal adhesions were observed in knockdown cells compared to controls.
Cell shape index discrepancies were noted between untreated and drug-treated cells.
Robust methodologies allowed for the analysis of large sample sizes, enhancing statistical relevance.

Conclusions

This study provides a reliable protocol for analyzing cell morphology and adhesion properties.
The findings contribute to understanding the mechanical properties of cells, relevant in cancer research and development.

Frequently Asked Questions

What are focal adhesions?

Focal adhesions are multiprotein complexes that mediate the attachment between the cell and the extracellular matrix, playing significant roles in cellular signaling and migration.

Why is MCF7 a relevant model for breast cancer research?

MCF7 cells are a widely studied human breast cancer cell line that exhibits many characteristics of primary breast tumors, making them valuable for cancer research.

How does the confocal microscopy technique improve image quality?

Confocal microscopy provides high-resolution images by using point illumination and spatially filtering out-of-focus light, enhancing the quality of the captured images.

What software is emphasized for image analysis in this study?

ImageJ is the primary software used for analyzing images to quantify focal adhesion area and cell shape index.

What are the key benefits of automated analysis described in the protocol?

Automated analysis facilitates the quantification of a large number of cells, increasing efficiency and accuracy in data collection.

Can this protocol be applied to other cell types?

Yes, the methodology can be adapted for various cell types, provided the parameters are appropriately established for each new cell line.

What are the implications of changes in cell shape index?

Alterations in cell shape index can indicate changes in cell behavior and functionality, relevant in cancer treatment responses and cellular mechanics.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

This protocol enables quantification of the area, perimeter, and shape of intracellular structure from its two-dimensional image. This technique is quick and easy, requires only standard laboratory settings and a confocal microscope and uses opensource software. Demonstrating the procedure will be Anna Balcerak, a PhD student from my laboratory.

Begin by seeding four times 10 to the fifth cells in 0.5 milliliters of culture medium per well in a four-well collagen one coated chamber slide for a 24-hour culture in a 37 degree Celsius and five degree carbon dioxide incubator. The next morning, use an optical inverted microscope to verify the presence of at least 90%confluent monolayer and use a confocal microscope to obtain single Z-slice images. For focal adhesion analysis, open the images in ImageJ and select analyze, set scale, remove scale, and global to set the image scale in pixels.

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