Abstract
BackgroundEffective tools for measurement of chemotaxis are desirable since cell migration towards given stimuli plays a crucial role in tumour metastasis, angiogenesis, inflammation, and wound healing. As for now, the Boyden chamber assay is the longstanding "gold-standard" for in vitro chemotaxis measurements. However, support for live cell microscopy is weak, concentration gradients are rather steep and poorly defined, and chemotaxis cannot be distinguished from migration in a single experiment.ResultsHere, we describe a novel all-in-one chamber system for long-term analysis of chemotaxis in vitro that improves upon many of the shortcomings of the Boyden chamber assay. This chemotaxis chamber was developed to provide high quality microscopy, linear concentration gradients, support for long-term assays, and observation of slowly migrating cells via video microscopy. AlexaFluor 488 dye was used to demonstrate the establishment, shape and time development of linear chemical gradients. Human fibrosarcoma cell line HT1080 and freshly isolated human umbilical vein endothelial cells (HUVEC) were used to assess chemotaxis towards 10% fetal calf serum (FCS) and FaDu cells' supernatant. Time-lapse video microscopy was conducted for 48 hours, and cell tracking and analysis was performed using ImageJ plugins. The results disclosed a linear steady-state gradient that was reached after approximately 8 hours and remained stable for at least 48 hours. Both cell types were chemotactically active and cell movement as well as cell-to-cell interaction was assessable.ConclusionsCompared to the Boyden chamber assay, this innovative system allows for the generation of a stable gradient for a much longer time period as well as for the tracking of cell locomotion along this gradient and over long distances. Finally, random migration can be distinguished from primed and directed migration along chemotactic gradients in the same experiment, a feature, which can be qualified via cell morphology imaging.
Highlights
Effective tools for measurement of chemotaxis are desirable since cell migration towards given stimuli plays a crucial role in tumour metastasis, angiogenesis, inflammation, and wound healing
Chemotaxis has been a focus of research for more than a century due to its involvement in several important physiological and pathological processes such as tumour metastasis [1,2], angiogenesis [3], inflammation [4], arteriosclerosis [5], and many other processes of great interest to biomedical research
Neo-angiogenesis is controlled by the production of chemotactic factors, which trigger the migration of endothelial cells into the tumour tissue
Summary
Effective tools for measurement of chemotaxis are desirable since cell migration towards given stimuli plays a crucial role in tumour metastasis, angiogenesis, inflammation, and wound healing. Migrating cells can be counted on the reverse side of the membrane after staining, usually as an endpoint assay at a predetermined time. These chemotaxis assays are widespread; the information obtained is limited, as live cell microscopy is substantially restricted, and gradients are very steep and rather undefined. This technique allows for the performance of many simultaneous assays in parallel, it has many limitations and drawbacks: In particular, the counting of migrated cells can be time consuming, tedious, and subject to error. The Boyden assays do not access the cells’paths or locomotion, and persistent chemotaxis cannot be distinguished from random migration in one single experiment; separate controls are required
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