En kvantitativ vurdering af Cell Migration af Phagokinetic Track Motilitet Assay

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 roles^1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread^4,5. 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 cell^6,7,8. 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 coverslip^9,10. 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 cells^11,12, fibroblasts⁹, neutrophils¹³, skeletal muscle cells¹⁴, keratinocytes¹⁵, trophoblasts¹⁶, endothelial cells¹⁷, and monocytes^10,18-22. 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 analyses^9,26,27.

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 analyzed^9-17, 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…

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.