JoVE Journal > Immunology and Infection
Summary
Abstract
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Immunology and Infection

A التقييم الكمي للهجرة الخليوي من قبل مصلحة دمغ المصوغات المسار على الحركة Phagokinetic

Published: December 04, 2012
doi:
10.3791/4165
, , , ,
1Department of Microbiology and Immunology,Louisiana State University Health Sciences Center, 2Center for Molecular and Tumor Virology,Louisiana State University Health Sciences Center, 3Department of Microbiology and Immunology,SUNY Upstate Medical University, 4Feist-Weiller Cancer Center,Louisiana State University Health Sciences Center

Summary

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Abstract

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.

The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.

The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au°) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.

In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

Protocol

1. Preparation of Gelatin-coated Coverslips Place acid-washed glass coverslips (15 mm in diameter) in a sterile plastic 100 mm dish(es). Place 8-9 coverslips per dish and make sure they are not touching each other or the sides of the dish.. Note: Coverslips, needles and tweezers need to be sterile to eliminate possible contaminating microorganisms, as well as endotoxins that will affect cellular functions, including motility. Weigh th…

Representative Results

Shown is an example of pictures taken under a light microscope showing a track area cleared by a single cell (a monocyte from our experiments is shown in Figure 2). Non-motile cells create characteristic small, oval or circle-shaped tracts around themselves indicating a low basal level of movement for these unstimulated cells (Figures 2A and 2B). In contrast, highly motile cells [in our system, human cytomegalovirus (HCMV)-infected cells] are characterized by a directional movement shown…

Discussion

The phagokinetic track motility assay presented in this article is a simple and highly effective method for quantitative analysis of cell migration. Because multiple cell types can be analyzed9-17, this method has the potential broad usage across multiple disciplines. The use of colloidal gold-coated glass coverslips allows for the measurement of a track area cleared by a moving cell. The assay can measure the effect of different stimuli (i.e. growth factors, purified ECM ligands, viruses, bac…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by grants from the National Institutes of Health (AI050677, HD-051998, and GM103433), a Malcolm Feist cardiovascular research fellowship, and an American Heart Association predoctoral fellowship (10PRE4200007).

Materials

Name of the reagent Company Catalog number Comments
Glass Coverslips (15 mm) Fisher Scientific 12-545-83  
Gelatin 300 Bloom Sigma-Aldrich G-1890  
Tetrachloroauric Acid Trihydrate Fisher Chemical G54-1 14.5 mM (a final working solution)
Sodium Citrate Fisher Scientific BP327-500 0.5% (a final working solution)
Paraformaldehyde Fisher Scientific O4042 3% (a final working solution)
100 mm Tissue Culture Dish Sarstedt 83.1802  
12-Well Plates Fisher Scientific 08-772-29  
24-Well Plates Fisher Scientific 07-200-84  
Techne Oven Hybridiser HB-1D LabPlanet 2040500 The standard laboratory oven will suffice
10 ml Serological Pipettes Sarstedt 86.1254.001  
Pipet-Aid Filler/Dispenser Drummond 13-681-15  
P200 Single-Channel Manual Pipette Rainin PR-200  
200 ml Barrier Tips CLP BT200  
ImageJ software http://rsb.info.nih.gov/ij/   License: Public Domain
Nikon Eclipse TE300 with a photometrics CoolSNAPfx monochrome 12-bit CCD camera Nikon   Discontinued; The most comparable specification has Nikon Eclipse Ti, but a lower end Nikon 80i will be suitable as well. Other brands also provide comparable microscopes.
      Note: The reagents and equipment listed below have been utilized by us in our various studies. Other supplies, suppliers, reagents, and equipment can be used, as long as they have similar specifications.

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References

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Tags

Cell MigrationPhagokinetic Track Motility AssayQuantitative EvaluationCellular MotilityUnicellular OrganismsMulticellular OrganismsImmune SurveillanceWound HealingNeurological DiseasesCardiovascular DiseasesImmunological DiseasesTumor FormationActin PolymerizationReceptor RecyclingCellular ExtensionsLamellipodiaCell MotilityMicroscopyQuantitative MeasurementResource-consuming Process

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Nogalski, M. T., Chan, G. C., Stevenson, E. V., Collins-McMillen, D. K., Yurochko, A. D. A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay. J. Vis. Exp. (70), e4165, doi:10.3791/4165 (2012).
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