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

Summary

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.

Abstract

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

Introduction

Epithelial cell monolayers act as a collective in which cell-cell and cell-substrate adhesion as well as contractile forces and tensions represent important parameters and their proper balance contributes to the overall integrity of the unit^1,2,3. Thus, assessing these parameters represents a way to establish the current status of the cell layer.

The two methods described here represent a two-dimensional analysis of the confluent monolayers of adherent, epithelial cells (in this case MCF7 breast cancer cell line). The analysis is performed using co....

Protocol

1. Preparatory steps

1. Cell seeding to obtain confluent monolayers
   1. Before seeding, coat the wells of a 4-wells chamber slide with collagen I (or other ECM component of choice). For collagen I coating, follow a commercial protocol: https://www.sigmaaldrich.com/technical-documents/articles/biofiles/collagen-product-protocols.html at a concentration of 8 μg/cm².
   2. Seed 400,000 cells to one well of a 4-well chamber slide.
   3. Culture the cells for 24 h (or longer, depending on the experimental endpoints) before staining, in an incubator at 37 °C, 5% CO₂. This step allows for a maturation of cell-cell contacts and....

Results

Focal adhesion analysis
The knockdown of HAX1 gene was previously shown to affect focal adhesions⁶. Cells were cultured on collagen I-coated surface for 48 h. Images of the MCF7 control cells and MCF7 cells with a HAX1 knockdown (HAX1 KD) from three independent experiments stained with focal adhesion protein paxillin were obtained using a confocal microscope (image from single focal plane/Z-slice from basal region). FAs from about 2,000-2,500 cells f.......

Discussion

Cell-cell and cell-substrate adhesion constitute inherent attributes of the epithelial cells and play the critical role in tissue morphogenesis and embriogenesis. In adult tissues the proper regulation of mechanical properties of the cell layer is crucial in maintaining homeostasis and preventing pathological responses like tumor progression and metastasis. The size and number of focal adhesions depend on the strength of cell-substrate adhesion, while cell shape depends on contractile forces and is related to the status .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 594	ThermoFisher Scientific	A32740	goat anti-rabbit, 1:500
Ammonium chloride	Sigma	A9434
BSA	BioShop	ALB001.500
Collagen from calf skin	Sigma	C9791-10MG
DAPI	Sigma	D9542	1:10000 (stock 1 mg/mL in H2O), nucleic acid staining
DMEM + GlutaMAX, 1 g/L D-Glucose, Pyruvate	ThermoFisher Scientific	21885-025
FBS	ThermoFisher Scientific	10270-136
Junction plakoglobin	Cell Signaling	2309S	rabbit, 1:400
Laminar-flow cabinet class 2	Alpina		standard equipment
MCF7-basedHAX1KD cell line	Cell line established in the National Institute of Oncology, Warsaw, described in Balcerak et al., 2019		MCF7 cell line withHAX1knockdown
MCF7 cell line (CONTROL)	ATCC	ATCC HTB-22	epithelial, adherent breast cancer cell line
Olympus CK2 light microscope	Olympus
Paxillin	Abcam	ab32084	rabbit, 1:250, Y113
PBS	ThermoFisher Scientific	10010023
Phalloidin-TRITC conjugate	Sigma	P1951	1:400 (stock 5 mg/mL in DMSO), actin labeling
PTX	Sigma	T7402-1MG
TBST – NaCl	Sigma	S9888
TBST – Trizma base	Sigma	T1503
Triton X-100	Sigma	9002-93-11
Zeiss LSM800 Confocal microscope	Zeiss