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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

Representative Results

A general schema of the experimental set-up

Figure 1 represents the general schema for the cell adhesion and spreading protocol beginning with serum starvation of REF52 cells and ending with computational analysis of acquired fluorescence images. Key steps in the protocol are illustrated in the timeline. Of note, step 2 of the protocol describes the preparation of the FN-coated coverslips, which should be performed concurrently with step 3: serum starving REF52 cell.......

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.......

Acknowledgements

The authors thank Drs. Scott R. Hutton and Meghan S. Blackledge for the critical review of the manuscript. This work was funded by High Point University’s Research and Sponsored Programs (MCS) and the Biotechnology Program at North Carolina State University (MCS).

....

Materials

NameCompanyCatalog NumberComments
0.05% Trypsin-EDTA (1x)Gibco by Life Technologies25300-054cell dissociation
10 cm2 dishesCell Treat229620sterile, tissue culture treated
15 mL conical tubesFisher Scientific05-539-5sterile
1X Phosphate Buffered SalineCorning Cellgro21-031-CVPBS, sterile, free of Mg2+ and Ca2+
24-well cell culture treated platesFisher Scientific07-200-740sterile, tissue culture treated
4°C refrigeratorFisher Scientific
Mouse IgG anti-paxillin primary antibody (clone 165)BD Transduction Laboratories610620marker of focal adhesions
AspiratorArgosEV310
Biosafety cabinetNuairNU-477-400Class II, Type A, series 5
Delipidated Bovine Serum Albumin (Fatty Acid Free) PowderFisher ScientificBP9704-100dlBSA
Dimethyl SulfoxideFisher ScientificBP231-100organic solvent to dissolve Ku55933
Dulbecco's Modified Eagle Media, High GlucoseFisher Scientific11965092REF52 base cell culture medium
Fetal bovine serumFisher Scientific16000044certified, cell culture medium supplement
FijiNational Institutes of Healthhttp://fiji.sc/image analysis program
Filter syringeFisher Scientific6900-25020.2 µM, sterile
Glass coverslips (12-Cir-1.5)Fisher Scientific12-545-81autoclave in foil to sterilize
Goat anti-mouse IgG secondary antibody Alexa Fluor 488InvitrogenA11001fluorescent secondary antibody, light sensitive
Goat SerumGibco by Life Technologies16210-064component of blocking solution for immunofluorescence
HemocytometerFisher Scientific22-600-107for cell counting
Human Plasma FibronectinGibco by Life Technologies33016-015FN
IX73 Fluorescence Inverted MicroscopeOlympusmicroscope to visualize fluorescence, cell morphology, counting and dissociation
Ku55933Sigma-AldrichSML1109-25MGATM kinase inhibitor, inducer of reactive oxygen species
L-glutamineFisher Scientific25-030-081cell culture medium supplement
Monochrome CMOS 16 bit cameraOptimos
ParaformaldehydeSigma-AldrichP6148-500GPFA, fixative for immunofluorescence
Penicillin-streptomycinFisher Scientific15-140-122P/S, antibiotic solution for culture medium
Alexa Fluor 594 phalloidin (F-actin probe)InvitrogenA12381marker of F-actin, light sensitive
ProLong Gold Anti-fade reagent with DAPIInvitrogenP36941cover slip mounting media including nuclear dye DAPI, light sensitive
REF52 cellsGraham, D.M. et. al. Journal of Cell Biology 2018
Stir plate with heat controlCorning IncorporatedPC-420D
SyringeBD Biosciences30965360 mL syringe
Tissue culture incubatorNuair
Triton X-100Fisher ScientificBP151-500detergent used to permeabilize cell membranes
Trypan Blue SolutionFisher Scientific15-250-061for cell counting
Trypsin Neutralizing Solution (1x)Gibco by Life TechnologiesR-002-100TNS, neutralizes trypsin instead of fetal bovine serum
tube rotatorFisher Scientific11-676-341
water bathFisher ScientificFSGPD02

References

  1. Geiger, B., Bershadsky, A., Pankov, R., Yamada, K. M. Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk. Nature Reviews: Molecular Cell Biology. 2 (11), 793-805 (2001).
  2. Geiger, B., Yamada, K. M.

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Cellular AdhesionCell SpreadingEpithelial CellsFibronectinOxidative StressExtracellular MatrixCytoskeletal DynamicsRedox StatusMetastatic CancerAnchorage dependent CellsCell CultureCell LinesOxidantsBSATrypsin EDTA

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