scholarly journals Vital Stain to Study Cell Invasion in Modified Boyden Chamber Assay

BioTechniques ◽  
2002 ◽  
Vol 33 (6) ◽  
pp. 1200-1204 ◽  
Author(s):  
Madhura R. Vipra ◽  
Jayant M. Chiplonkar
Keyword(s):  
Cell Invasion ◽  
Boyden Chamber ◽  
Vital Stain ◽  
Boyden Chamber Assay

scholarly journals Modification of the Boyden Chamber to Improve Uniformity of Cell Invasion of Matrigel-Coated Membranes

BioTechniques ◽  
2001 ◽  
Vol 31 (6) ◽  
pp. 1242-1246 ◽  
Author(s):  
Kelly L. Bloomfield ◽  
Ben L. Baldwin ◽  
Damien G. Harkin ◽  
Kathryn F. Tonissen
Keyword(s):  
Cell Invasion ◽  
Boyden Chamber ◽  
Coated Membranes

scholarly journals MMP3 activity rather than cortical stiffness determines NHE1-dependent invasiveness of melanoma cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Dennis Keurhorst ◽  
Ivan Liashkovich ◽  
Fabian Frontzek ◽  
Svenja Nitzlaff ◽  
Verena Hofschröer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Invasion ◽  
Time Lapse ◽  
Actin Dynamics ◽  
Cell Stiffness ◽  
Migration And Invasion ◽  
Boyden Chamber ◽  
Cortical Actin ◽  
Structural Element ◽  
Mmp Inhibitor

Abstract Background Both cell adhesion and matrix metalloproteinase (MMP) activity depend on pH at the cell surface. By regulating extracellular juxtamembrane pH, the Na+/H+ exchanger NHE1 plays a significant part in human melanoma (MV3) cell migration and invasion. Because NHE1, besides its pH-regulatory transport function, also serves as a structural element tying the cortical actin cytoskeleton to the plasma membrane, we investigated whether NHE1 affects cortical stiffness of MV3 cells, and how this makes an impact on their invasiveness. Methods NHE1 overexpressing MV3 cells were compared to the corresponding mock-transfected control cells. NHE1 expression was verified by Western blotting, cariporide (HOE642) was used to inhibit NHE1 activity, cell stiffness was determined by atomic force microscopy, and F-actin was visualized by phalloidin-staining. Migration on, and invasion of, native and glutaraldehyde-fixed collagen I substrates were analyzed using time-lapse video microscopy and Boyden-chamber assays, respectively. MMP secretion and activity were detected by Western blot and zymography, respectively. MMP activity was inhibited with NNGH. Results The cortical, but not the bulk stiffness, was significantly higher in NHE1 overexpressing cells. This increase in cortical stiffness was accompanied by a reorganization of the cortical cytoskeleton, i.e. a condensation of F-actin underneath and along the plasma membrane. However, it was not affected by NHE1 inhibition. Nevertheless, actin dynamics is required for cell invasion as demonstrated with the application of cytochalasin D. NHE1 overexpression was associated with an elevated MMP3 secretion and an increase in the invasion of a native matrix. This increase in invasiveness could be antagonized by the MMP inhibitor NNGH. Transmigration through a glutaraldehyde-fixed, indigestible substrate was not affected by NHE1 overexpression. Conclusion NHE1, as a structural element and independently of its transport activity, contributes to the organization of the cortical F-actin meshwork and thus impacts cortical stiffness. Since NHE1 overexpression stimulates MMP3 secretion but does not change transmigration through a fixed substrate, MV3 cell invasion of a native substrate depends on MMP activity rather than on a modifiable cortical stiffness.

scholarly journals Prodigiosin Inhibits Proliferation, Migration, and Invasion of Nasopharyngeal Cancer Cells

2018 ◽  
Vol 48 (4) ◽  
pp. 1556-1562 ◽  
Author(s):  
Yongze Liu ◽  
Han Zhou ◽  
Xiaofeng Ma ◽  
Chuanyao Lin ◽  
Ling Lu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Nasopharyngeal Carcinoma ◽  
Tumor Cell ◽  
Cancer Cells ◽  
Cell Invasion ◽  
Nasopharyngeal Cancer ◽  
Migration And Invasion ◽  
Boyden Chamber ◽  
Cell Cycle Distribution ◽  
Tumor Cell Migration

Background/Aims: Nasopharyngeal carcinoma remains a devastating and difficult disease to treat. This study explores the antineoplastic effect of prodigiosin on nasopharyngeal cancer cells. Methods: Human nasopharyngeal carcinoma CNE2 cells and human normal nasopharyngeal epithelial NP69 cells were obtained and treated with prodigiosin or fluorouracil (5-FU). Colony formation assay was performed to screen for the optimal experimental concentrations of prodigiosin and 5-FU, and MTT assay was used to examine cell proliferative ability. Flow cytometry was used to examine cell cycle distribution, the scratch test was employed to examine cell migration, and Transwell migration assay (Boyden chamber) was used to study cell invasion. Results: The optimal concentrations of prodigiosin and 5-FU for treatment were 4 mg/L and 0.35 mg/L, respectively. Both prodigiosin and 5-FU inhibited tumor cell proliferation. The percentage of cells in G0/G1 phase was higher and the percentage of cells in S phase was lower in the prodigiosin and 5-FU groups than in the untreated groups. Both prodigiosin and 5-FU inhibited tumor cell migration and tumor cell invasion. Conclusions: Our results suggest that prodigiosin can inhibit proliferation, migration, and invasion of nasopharyngeal carcinoma cells.

CCL-2 Is a KIT D816V-Dependent Modulator of Bone Marrow Remodeling and Microenvironmental Alterations in Systemic Mastocytosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1635-1635
Author(s):  
Georg Greiner ◽  
Nadine Witzeneder ◽  
Klaus Schmetterer ◽  
Theresia Popow-Kraupp ◽  
Wolfgang R Sperr ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conditioned Medium ◽  
Research Funding ◽  
Systemic Mastocytosis ◽  
Tissue Remodeling ◽  
Neutralizing Antibody ◽  
Boyden Chamber ◽  
Boyden Chamber Assay ◽  
Baseline Levels ◽  
Kit D816v

Abstract Systemic mastocytosis (SM) is a myeloproliferative neoplasm (MPN) characterized by mast cell (MC) infiltration in the bone marrow (BM) and/or other organs. Most patients present with the activating KIT mutation D816V. Increased production of cytokines is typically found in patients with classical MPN and has been linked to alterations of the BM microenvironment (e.g. enhanced angiogenesis and fibrosis) that are also commonly found in SM. However, little is known about mechanisms contributing to microenvironment alterations in the BM in SM and the role of MC-derived cytokines. The aim of our study was to identify cytokines that are produced in neoplastic MC in a KIT-dependent manner and contribute to increased BM angiogenesis and fibrosis in SM. In a first step, we screened for KIT D816V-dependent production of cytokines relevant to inflammation and microenvironment alterations using growth factor-dependent human cell lines (TF-1 and Mo7e). In these experiments, expression of CCL-2, IL-8, OSM, and VEGF mRNA was induced by KIT D816V but not by wild type KIT. Based on its pleiotropic effects, we focused on CCL-2, also referred to as monocyte chemotactic protein 1 (MCP-1), a CC chemokine that recruits inflammatory cells to sites of inflammation and enhances angiogenesis. KIT D816V+ HMC-1.2 cell were found to express and secrete substantial amounts of CCL-2. Midostaurin, a multikinase inhibitor suppressing the kinase activity of KIT D816V, was found to reduce expression of CCL-2 similar to RNAi-mediated knockdown of KIT. Furthermore, knockdown of STAT5, a key transcription factor downstream of KIT D816V, reduced expression of CCL-2 in HMC-1.2 cells. These results confirmed that CCL-2 expression in neoplastic MC is dependent on KIT D816V. Since CCL-2 has been reported to promote angiogenesis, we analyzed effects of conditioned medium obtained from HMC-1.2 cells on human umbilical vein endothelial cells (HUVEC) known to express the CCL-2 receptor CCR-2. Indeed, MC-derived conditioned medium induced migration of HUVEC in a wound healing assay (214% ± 38% of control, mean ± SD, p<0.01) as well as in a boyden chamber assay (126% ± 8% of control, p<0.05). Moreover, pre-incubation with a neutralizing antibody against CCL-2 significantly reduced migratory responses (p<0.01), and RNAi-mediated knockdown of CCL-2 in neoplastic MC reduced the effect of conditioned medium to baseline levels (p<0.01). These results confirmed that the migration was CCL-2 dependent. Furthermore, patients with advanced SM often present with marked eosinophilia; and eosinophils are often located in the vicinity of, or even within, BM MC infiltrates. Therefore, we also studied the effect of CCL-2 on eosinophils. Normal human eosinophils as well as the eosinophilic cell line EOL-1 were found to express CCR-2, and to migrate against recombinant CCL-2 in a modified boyden chamber assay. Conditioned medium from KIT D816V+ MC also induced a migratory response in eosinophils (200% ± 30% of control, p<0.05), and a neutralizing antibody against CCL-2 reduced this effect to baseline levels (p<0.05). Finally, SM-patients were found to have significantly elevated serum CCL-2 levels (n=35, p=0.0048, 407.4 ± 42.3 pg/ml, mean ± SEM) compared to controls (274.3 ± 15.9 pg/ml). The highest serum levels of CCL-2 were detected in patients with advanced SM where tissue remodeling in the BM is often a prominent feature. Moreover, CCL-2 levels were found to correlate with the grade of MC infiltration in the BM (r=0.656, p=0.0002). In summary, KIT D816V induces the expression of various cytokines potentially involved in tissue remodeling and microenvironment alterations in SM. Moreover, we have identified CCL-2 as a critical, KIT D816V-dependent, cytokine-mediator that interacts with structural BM cells and thereby may be involved in disease evolution and progression in SM. Whether CCL-2 may also serve as a therapeutic target in SM is currently being examined. This study was supported by Austrian Science Fund (FWF) grant P26079-B13. Disclosures Sperr: Novartis: Honoraria. Valent:Pfizer: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

A simplified Boyden chamber assay for neutrophil chemotaxis based on quantitation of myeloperoxidase

1990 ◽  
Vol 185 (2) ◽  
pp. 238-242 ◽  
Author(s):  
Kristina Somersalo ◽  
Osmo P. Salo ◽  
Fred Björkstén ◽  
Kimmo K. Mustakallio
Keyword(s):  
Boyden Chamber ◽  
Neutrophil Chemotaxis ◽  
Boyden Chamber Assay
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