scholarly journals In vitro cell migration quantification method for scratch assays

2019 ◽  
Vol 16 (151) ◽  
pp. 20180709 ◽  
Author(s):  
Ana Victoria Ponce Bobadilla ◽  
Jazmine Arévalo ◽  
Eduard Sarró ◽  
Helen M. Byrne ◽  
Philip K. Maini ◽  
...  
Keyword(s):  
Experimental Data ◽  
Cell Migration ◽  
Cell Monolayer ◽  
Leading Edge ◽  
New Method ◽  
Scratch Assay ◽  
Standard Methods ◽  
Migration Rates ◽  
Quantification Method

The scratch assay is an in vitro technique used to assess the contribution of molecular and cellular mechanisms to cell migration. The assay can also be used to evaluate therapeutic compounds before clinical use. Current quantification methods of scratch assays deal poorly with irregular cell-free areas and crooked leading edges which are features typically present in the experimental data. We introduce a new migration quantification method, called ‘monolayer edge velocimetry’, that permits analysis of low-quality experimental data and better statistical classification of migration rates than standard quantification methods. The new method relies on quantifying the horizontal component of the cell monolayer velocity across the leading edge. By performing a classification test on in silico data, we show that the method exhibits significantly lower statistical errors than standard methods. When applied to in vitro data, our method outperforms standard methods by detecting differences in the migration rates between different cell groups that the other methods could not detect. Application of this new method will enable quantification of migration rates from in vitro scratch assay data that cannot be analysed using existing methods.

scholarly journals CaM kinase IIδ2-dependent regulation of vascular smooth muscle cell polarization and migration

2008 ◽  
Vol 294 (6) ◽  
pp. C1465-C1475 ◽  
Author(s):  
Melissa Z. Mercure ◽  
Roman Ginnan ◽  
Harold A. Singer
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Vascular Smooth Muscle ◽  
Cell Monolayer ◽  
Leading Edge ◽  
Cell Polarization ◽  
Loss Of Function ◽  
Downstream Signaling ◽  
Transient Activation ◽  
And Migration

Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKIIδ2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKIIδ2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKIIδ2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKIIδ2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKIIδ2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

scholarly journals Role of collagenase in mediating in vitro alveolar epithelial wound repair

1999 ◽  
Vol 112 (2) ◽  
pp. 243-252
Author(s):  
E. Planus ◽  
S. Galiacy ◽  
M. Matthay ◽  
V. Laurent ◽  
J. Gavrilovic ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Adhesion ◽  
Epithelial Cell ◽  
Type I Collagen ◽  
Alveolar Epithelial Cell ◽  
Cell Monolayer ◽  
Type I ◽  
Type Ii ◽  
Alveolar Epithelial

Type II pneumocytes are essential for repair of the injured alveolar epithelium. The effect of two MMP collagenases, MMP-1 and MMP-13 on alveolar epithelial repair was studied in vitro. The A549 alveolar epithelial cell line and primary rat alveolar epithelial cell cultures were used. Cell adhesion and cell migration were measured with and without exogenous MMP-1. Wound healing of a cell monolayer of rat alveolar epithelial cell after a mechanical injury was evaluated by time lapse video analysis. Cell adhesion on type I collagen, as well as cytoskeleton stiffness, was decreased in the presence of exogenous collagenases. A similar decrease was observed when cell adhesion was tested on collagen that was first incubated with MMP-1 (versus control on intact collagen). Cell migration on type I collagen was promoted by collagenases. Wound healing of an alveolar epithelial cell monolayer was enhanced in the presence of exogenous collagenases. Our results suggest that collagenases could modulate the repair process by decreasing cell adhesion and cell stiffness, and by increasing cell migration on type I collagen. Collagen degradation could modify cell adhesion sites and collagen degradation peptides could induce alveolar type II pneumocyte migration. New insights regarding alveolar epithelial cell migration are particularly relevant to investigate early events during alveolar epithelial repair following lung injury.

scholarly journals A particle-based model for endothelial cell migration under flow conditions

2019 ◽  
Vol 19 (2) ◽  
pp. 681-692
Author(s):  
P. S. Zun ◽  
A. J. Narracott ◽  
P. C. Evans ◽  
B. J. M. van Rooij ◽  
A. G. Hoekstra
Keyword(s):  
Experimental Data ◽  
Cell Migration ◽  
Flow Direction ◽  
Healing Process ◽  
Endothelial Cell Migration ◽  
Bottom Surface ◽  
Flow Conditions ◽  
Directed Movement ◽  
Local Flow

Abstract Endothelial cells (ECs) play a major role in the healing process following angioplasty to inhibit excessive neointima. This makes the process of EC healing after injury, in particular EC migration in a stented vessel, important for recovery of normal vessel function. In that context, we present a novel particle-based model of EC migration and validate it against in vitro experimental data. We have developed a particle-based model of EC migration under flow conditions in an in vitro vessel with obstacles. Cell movement in the model is a combination of random walks and directed movement along the local flow velocity vector. For model calibration, a set of experimental data for cell migration in a similarly shaped channel has been used. We have calibrated the model for a baseline case of a channel with no obstacles and then applied it to the case of a channel with ridges on the bottom surface, representative of stent strut geometry. We were able to closely reproduce the cell migration speed and angular distribution of their movement relative to the flow direction reported in vitro. The model also reproduces qualitative aspects of EC migration, such as entrapment of cells downstream from the flow-disturbing ridge. The model has the potential, after more extensive in vitro validation, to study the effect of variation in strut spacing and shape, through modification of the local flow, on EC migration. The results of this study support the hypothesis that EC migration is strongly affected by the direction and magnitude of local wall shear stress.

scholarly journals A Quantitative, Facile, and High-Throughput Image-Based Cell Migration Method Is a Robust Alternative to the Scratch Assay

2011 ◽  
Vol 16 (2) ◽  
pp. 155-163 ◽  
Author(s):  
Wendy Gough ◽  
Keren I. Hulkower ◽  
Renee Lynch ◽  
Patrick Mcglynn ◽  
Mark Uhlik ◽  
...  
Keyword(s):  
Cell Migration ◽  
Kinase Inhibitor ◽  
Cell Monolayer ◽  
Cost Effective ◽  
Statistical Validation ◽  
Scratch Assay ◽  
Cell Migration Assay ◽  
Migration Assay ◽  
Confluent Cell ◽  
Releasing Factors

Cell migration is a key phenotype for a number of therapeutically important biological responses, including angiogenesis. A commonly used method to assess cell migration is the scratch assay, which measures the movement of cells into a wound made by physically scoring a confluent cell monolayer to create an area devoid of cells. Although this method has been adequate for qualitative characterization of migration inhibitors, it does not provide the highly reproducible results required for quantitative compound structure-activity relationship evaluation because of the inconsistent size and placement of the wound area within the microplate well. The Oris™ Cell Migration Assay presents a superior alternative to the scratch assay, permitting formation of precisely placed and homogeneously sized cell-free areas into which migration can occur without releasing factors from wounded or dead cells or damaging the underlying extracellular matrix. Herein the authors compare results from the scratch and Oris™ cell migration assays using an endothelial progenitor cell line and the Src kinase inhibitor dasatinib. They find that using the Acumen™ Explorer laser microplate cytometer in combination with the Oris™ Cell Migration Assay plate provides a robust, efficient, and cost-effective cell migration assay exhibiting excellent signal to noise, plate uniformity, and statistical validation metrics.

scholarly journals Modelling GTPase dynamics to understand RhoA-driven cancer cell invasion

2016 ◽  
Vol 44 (6) ◽  
pp. 1695-1700 ◽  
Author(s):  
Joseph H.R. Hetmanski ◽  
Jean-Marc Schwartz ◽  
Patrick T. Caswell
Keyword(s):  
Cell Migration ◽  
Rho Gtpases ◽  
Leading Edge ◽  
Local Environment ◽  
Future Research ◽  
Rhoa Activity ◽  
Rac1 Activity ◽  
Logical Modelling ◽  
Invasive Cell

Metastasis, initially driven by cells migrating and invading through the local environment, leads to most cancer-associated deaths. Cells can use a variety of modes to move in vitro, all of which depend on Rho GTPases at some level. While traditionally it was thought that Rac1 activity drives protrusive lamellipodia at the leading edge of a polarised cell while RhoA drives rear retraction, more recent work in 3D microenvironments has revealed a much more complicated picture of GTPase dynamics. In particular, RhoA activity can dominate the leading edge polymerisation of actin to form filopodial actin-spike protrusions that drive more invasive cell migration. We recently described a potential mechanism to abrogate this pro-invasive localised leading edge Rac1 to RhoA switch via manipulation of a negative feedback loop that was revealed by adopting a logical modelling approach. Both challenging dogma and taking a formal, mathematical approach to understanding signalling involved in motility may be vital to harnessing harmful cell migration and preventing metastasis in future research.

scholarly journals α4/α9 Integrins Coordinate Epithelial Cell Migration Through Local Suppression of MAP Kinase Signaling Pathways

2021 ◽  
Vol 9 ◽  
Author(s):  
Willow Hight-Warburton ◽  
Robert Felix ◽  
Andrew Burton ◽  
Hannah Maple ◽  
Magda S. Chegkazi ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Complexes ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Collective Cell Migration ◽  
Keratinocyte Proliferation ◽  
Basal Keratinocytes ◽  
Keratinocyte Cell

Adhesion of basal keratinocytes to the underlying extracellular matrix (ECM) plays a key role in the control of skin homeostasis and response to injury. Integrin receptors indirectly link the ECM to the cell cytoskeleton through large protein complexes called focal adhesions (FA). FA also function as intracellular biochemical signaling platforms to enable cells to respond to changing extracellular cues. The α4β1 and α9β1 integrins are both expressed in basal keratinocytes, share some common ECM ligands, and have been shown to promote wound healing in vitro and in vivo. However, their roles in maintaining epidermal homeostasis and relative contributions to pathological processes in the skin remain unclear. We found that α4β1 and α9β1 occupied distinct regions in monolayers of a basal keratinocyte cell line (NEB-1). During collective cell migration (CCM), α4 and α9 integrins co-localized along the leading edge. Pharmacological inhibition of α4β1 and α9β1 integrins increased keratinocyte proliferation and induced a dramatic change in cytoskeletal remodeling and FA rearrangement, detrimentally affecting CCM. Further analysis revealed that α4β1/α9β1 integrins suppress extracellular signal-regulated kinase (ERK1/2) activity to control migration through the regulation of downstream kinases including Mitogen and Stress Activated Kinase 1 (MSK1). This work demonstrates the roles of α4β1 and α9β1 in regulating migration in response to damage cues.

In vitro studies in Tubularia: a morphological role for digestive cells

Development ◽  
1973 ◽  
Vol 29 (1) ◽  
pp. 1-13
Author(s):  
Marc H. Glickman ◽  
Georgia E. Lesh-Laurie
Keyword(s):  
Interstitial Cell ◽  
Cell Attachment ◽  
Cell Monolayer ◽  
Leading Edge ◽  
Late Summer ◽  
Autoradiographic Study ◽  
Digestive Cell ◽  
Digestive Cells ◽  
And Migration

A chemically defined in vitro system for Tubularia has been developed. At 24 h after explantation of coenosarc, digestive cells attached to the substrate and migrated from the explant. The digestive cells migrated by a gliding motion with a fan-like membrane acting as a leading edge. Within 48 h a digestive cell monolayer was formed and invasion of this area by epithelio-muscular, gland and interstitial cells occurred. Autoradiographic study of 48–72 h cultures treated with [3H]thymidine showed nuclear incorporation of the label in digestive, epithelio-muscular, interstitial, cnidoblast and gland cells. When explants were grown on collagen-coated coverslips, accelerated attachment and migration of digestive cells was observed. Explants were also grown on Millipore filters. No digestive cell attachment occurred but epithelio-muscular, gland and interstitial cell attachments to the filter were observed. From these experiments, a morphological role for the digestive cells as a substrate for other cells of the coenosarc is postulated. Hydranth extract was supplemented to the culture medium. Studies with this material were performed with coenosarc from ‘late summer’ animals in which only 10–15% of the explants normally entered culture. However, with the addition of the extract, 100% of the explants went into culture. Interstitial cell populations increased 2–3 times in extract-treated explants.

scholarly journals Cytotoxicity evaluation of chlorhexidine gluconate on human fibroblasts, myoblasts, and osteoblasts

2018 ◽  
Vol 3 (4) ◽  
pp. 165-172 ◽  
Author(s):  
James X. Liu ◽  
Jordan Werner ◽  
Thorsten Kirsch ◽  
Joseph D. Zuckerman ◽  
Mandeep S. Virk
Keyword(s):  
Cell Migration ◽  
Cell Survival ◽  
Human Fibroblasts ◽  
Cell Monolayer ◽  
Survival Rates ◽  
Chlorhexidine Gluconate ◽  
Cell Counting ◽  
In Vivo Studies ◽  
Wound Irrigation

Abstract. Introduction: Chlorhexidine gluconate (CHX) is widely used as a preoperative surgical skin-preparation solution and intra-wound irrigation agent, with excellent efficacy against wide variety of bacteria. The cytotoxic effect of CHX on local proliferating cells following orthopaedic procedures is largely undescribed. Our aim was to investigate the in vitro effects of CHX on primary fibroblasts, myoblasts, and osteoblasts.Methods: Cells were exposed to CHX dilutions (0%, 0.002%, 0.02%, 0.2%, and 2%) for either a 1, 2, or 3-minute duration. Cell survival was measured using a cytotoxicity assay (Cell Counting Kit-8). Cell migration was measured using a scratch assay: a “scratch” was made in a cell monolayer following CHX exposure, and time to closure of the scratch was measured.Results: All cells exposed to CHX dilutions of ≥ 0.02% for any exposure duration had cell survival rates of less than 6% relative to untreated controls (p < 0.001). Cells exposed to CHX dilution of 0.002% all had significantly lower survival rates relative to control (p < 0.01) with the exception of 1-minute exposure to fibroblasts, which showed 96.4% cell survival (p = 0.78). Scratch defect closure was seen in < 24 hours in all control conditions. However, cells exposed to CHX dilutions ≥ 0.02% had scratch defects that remained open indefinitely.Conclusions: The clinically used concentration of CHX (2%) permanently halts cell migration and significantly reduces survival of in vitro fibroblasts, myoblasts, and osteoblasts. Further in vivo studies are required to examine and optimize CHX safety and efficacy when applied near open incisions or intra-wound application.

scholarly journals Promotion of Keratinocyte Proliferation by Tracheloside through ERK1/2 Stimulation

2018 ◽  
Vol 2018 ◽  
pp. 1-5 ◽  
Author(s):  
JaeGoo Kim ◽  
Yu-Kyong Shin ◽  
Ki-Young Kim
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Positive Control ◽  
Skin Injury ◽  
Scratch Assay ◽  
Keratinocyte Proliferation ◽  
Promote Cell Migration ◽  
A Cell ◽  
Cell Migration And Proliferation

Cell migration and proliferation are important for proper wound healing after skin injury. Recent studies have shown that compounds from plants could promote cell migration and proliferation. Tracheloside, which is a plant lignan, has been found to promote the growth of HaCaT cells over 40% compared to other compounds tested based on a cell proliferation assay. An in vitro scratch assay confirmed the healing activity of tracheloside (more than 2-fold increased healing activity after 24 hours of treatment compared with the control) and revealed that this activity is better than that of allantoin (1.2-fold increased after 24 hours of treatment compared with the control), a positive control. With western blot results, wound healing with tracheloside occurred through the phosphorylation of ERK1/2. Therefore, tracheloside is a good candidate to promote wound healing and could be developed as a therapeutic agent for wound treatment or used as a leading compound with higher activity.

scholarly journals Airway Epithelial Cell Migration Dynamics: MMP-9 Role in Cell–Extracellular Matrix Remodeling

1999 ◽  
Vol 146 (2) ◽  
pp. 517-529 ◽  
Author(s):  
Claire Legrand ◽  
Christine Gilles ◽  
Jean-Marie Zahm ◽  
Myriam Polette ◽  
Anne-Cécile Buisson ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Leading Edge ◽  
Collagen Iv ◽  
Matrix Remodeling ◽  
Airway Epithelial ◽  
Type Iv ◽  
Migration Dynamics

Cell spreading and migration associated with the expression of the 92-kD gelatinase (matrix metalloproteinase 9 or MMP-9) are important mechanisms involved in the repair of the respiratory epithelium. We investigated the location of MMP-9 and its potential role in migrating human bronchial epithelial cells (HBEC). In vivo and in vitro, MMP-9 accumulated in migrating HBEC located at the leading edge of a wound and MMP-9 expression paralleled cell migration speed. MMP-9 accumulated through an actin-dependent pathway in the advancing lamellipodia of migrating cells and was subsequently found active in the extracellular matrix (ECM). Lamellipodia became anchored through primordial contacts established with type IV collagen. MMP-9 became amassed behind collagen IV where there were fewer cell–ECM contacts. Both collagen IV and MMP-9 were involved in cell migration because when cell–collagen IV interaction was blocked, cells spread slightly but did not migrate; and when MMP-9 activation was prevented, cells remained fixed on primordial contacts and did not advance at all. These observations suggest that MMP-9 controls the migration of repairing HBEC by remodeling the provisional ECM implicated in primordial contacts.

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