A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

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 10,[18][19][20][21][22] . The protocol involves the creation of slides coated with gold nanoparticles (Au°) that are generated by a reduction of chloroauric acid (Au 3+ ) by sodium citrate. This method was developed by Turkevich et al. in 1951 23 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 sc...

Human Cytomegalovirus Utilizes a Nontraditional Signal Transducer and Activator of Transcription 1 Activation Cascade via Signaling through Epidermal Growth Factor Receptor and Integrins To Efficiently Promote the Motility, Differentiation, and Polarization of Infected Monocytes

“…Glass coverslips coated with colloidal gold were prepared as previously described (51,87). Monocytes were transfected with STAT1-specific or nonspecific control siRNA and incubated in suspension at 37°C for 24 h. Monocytes were then mock or HCMV infected and plated on colloidal gold-coated coverslips at a cell density of 5 ϫ 10 4 monocytes/coverslip.…”

“…6A). We utilized our monocyte motility assay (51,87) to compare the relative motility of untransfected monocytes and monocytes transfected with control siRNA with that of monocytes transfected with STAT1-specific siRNA…”

“…Because we previously showed that HCMV-infected monocytes undergoing monocyte-to-macrophage differentiation possess a survival advantage over their uninfected, nondifferentiating counterparts (12,21,30,35; reviewed in reference 31), we next asked whether the presence of STAT1 affected the survival of monocytes during infection. We performed 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays to compare the survival rates of uninfected and (87) and incubated at 37°C. At 24 hpi, images of individual monocytes were taken at a magnification of ϫ200 under bright-field microscopy.…”

CPEB1 promotes differentiation and suppresses EMT in mammary epithelial cells

“…We tested whether CPEB1 knockdown changes the motile properties of CID-9 cells with a phagokinetic track motility assay (Nogalski et al, 2012). In this assay, a cell moving on a colloidal-gold-coated glass coverslip clears gold particles and creates a measurable tract.…”

“…Phagokinetic track motility assay CID-9 cells (10 3 ) were transferred to colloidal-gold-coated coverslips (Nogalski et al, 2012) in 12-well plates and kept for 18 h at 37˚C under 5% CO 2 . Individual images were captured, and the average area of colloidal gold cleared by at least ten cells was determined for each well with ImageJ software (Abramoff et al, 2004).…”