Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

Summary

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

Abstract

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

Introduction

Cell-matrix adhesions (i.e., focal adhesions) are large and dynamic multimolecular protein complexes which mediate cell adhesion and spreading. These processes are critical for tissue development, maintenance, and physiological function. Focal adhesions are composed of membrane-bound receptors, such as integrins, as well as scaffolding proteins that link cytoskeletal actin to the extracellular matrix (ECM)¹. These complexes are capable of responding to physiochemical cues present in the extracellular environment through the activation of various signaling transduction pathways. As such, focal adhesions serve as signaling centers to propagate ex....

Protocol

1. Preparations

NOTE: The protocol described below has been optimized for the use with REF52 cells and ATM^+/+ or ATM^-/- human fibroblasts. Other cell types may require further optimization as described in the notes and troubleshooting sections below.

1. Make 500 mL of complete cell culture medium for REF52 cells. To 500 mL of high-glucose containing Dulbecco’s modified Eagle’s medium (DMEM) add 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin-st.......

Discussion

The protocol described here is a versatile and economical way to rapidly screen a number of anchorage-dependent cell types for dynamic cytoskeleton remodeling during cell spreading. In particular, this method quantitatively examines stress fiber and focal adhesion formation during oxidative stress when cells adhere to FN (Figure 1A). Moreover, these cellular phenotypes may suggest a regulatory role for members of the Rho family of small GTPases since they have documented roles during cell at.......

Materials

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA (1x)	Gibco by Life Technologies	25300-054	cell dissociation
10 cm2 dishes	Cell Treat	229620	sterile, tissue culture treated
15 mL conical tubes	Fisher Scientific	05-539-5	sterile
1X Phosphate Buffered Saline	Corning Cellgro	21-031-CV	PBS, sterile, free of Mg2+ and Ca2+
24-well cell culture treated plates	Fisher Scientific	07-200-740	sterile, tissue culture treated
4°C refrigerator	Fisher Scientific
Mouse IgG anti-paxillin primary antibody (clone 165)	BD Transduction Laboratories	610620	marker of focal adhesions
Aspirator	Argos	EV310
Biosafety cabinet	Nuair	NU-477-400	Class II, Type A, series 5
Delipidated Bovine Serum Albumin (Fatty Acid Free) Powder	Fisher Scientific	BP9704-100	dlBSA
Dimethyl Sulfoxide	Fisher Scientific	BP231-100	organic solvent to dissolve Ku55933
Dulbecco's Modified Eagle Media, High Glucose	Fisher Scientific	11965092	REF52 base cell culture medium
Fetal bovine serum	Fisher Scientific	16000044	certified, cell culture medium supplement
Fiji	National Institutes of Health	http://fiji.sc/	image analysis program
Filter syringe	Fisher Scientific	6900-2502	0.2 µM, sterile
Glass coverslips (12-Cir-1.5)	Fisher Scientific	12-545-81	autoclave in foil to sterilize
Goat anti-mouse IgG secondary antibody Alexa Fluor 488	Invitrogen	A11001	fluorescent secondary antibody, light sensitive
Goat Serum	Gibco by Life Technologies	16210-064	component of blocking solution for immunofluorescence
Hemocytometer	Fisher Scientific	22-600-107	for cell counting
Human Plasma Fibronectin	Gibco by Life Technologies	33016-015	FN
IX73 Fluorescence Inverted Microscope	Olympus		microscope to visualize fluorescence, cell morphology, counting and dissociation
Ku55933	Sigma-Aldrich	SML1109-25MG	ATM kinase inhibitor, inducer of reactive oxygen species
L-glutamine	Fisher Scientific	25-030-081	cell culture medium supplement
Monochrome CMOS 16 bit camera	Optimos
Paraformaldehyde	Sigma-Aldrich	P6148-500G	PFA, fixative for immunofluorescence
Penicillin-streptomycin	Fisher Scientific	15-140-122	P/S, antibiotic solution for culture medium
Alexa Fluor 594 phalloidin (F-actin probe)	Invitrogen	A12381	marker of F-actin, light sensitive
ProLong Gold Anti-fade reagent with DAPI	Invitrogen	P36941	cover slip mounting media including nuclear dye DAPI, light sensitive
REF52 cells			Graham, D.M. et. al. Journal of Cell Biology 2018
Stir plate with heat control	Corning Incorporated	PC-420D
Syringe	BD Biosciences	309653	60 mL syringe
Tissue culture incubator	Nuair
Triton X-100	Fisher Scientific	BP151-500	detergent used to permeabilize cell membranes
Trypan Blue Solution	Fisher Scientific	15-250-061	for cell counting
Trypsin Neutralizing Solution (1x)	Gibco by Life Technologies	R-002-100	TNS, neutralizes trypsin instead of fetal bovine serum
tube rotator	Fisher Scientific	11-676-341
water bath	Fisher Scientific	FSGPD02