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A novel chemotaxis assay in 3-D collagen gels by time-lapse microscopy.

Vasaturo A, Caserta S, Russo I, Preziosi V, Ciacci C, Guido S - PLoS ONE (2012)

Bottom Line: The results are expressed in terms of chemotactic index (I) and average cell velocity.These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix.The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Ingegneria Chimica Università di Napoli Federico II, Naples, Italy.

ABSTRACT
The directional cell response to chemical gradients, referred to as chemotaxis, plays an important role in physiological and pathological processes including development, immune response and tumor cell invasion. Despite such implications, chemotaxis remains a challenging process to study under physiologically-relevant conditions in-vitro, mainly due to difficulties in generating a well characterized and sustained gradient in substrata mimicking the in-vivo environment while allowing dynamic cell imaging. Here, we describe a novel chemotaxis assay in 3D collagen gels, based on a reusable direct-viewing chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane. The diffusion process has been analysed by monitoring the concentration of FITC-labelled dextran through epifluorescence microscopy and by comparing experimental data with theoretical and numerical predictions based on Fick's law. Cell migration towards chemoattractant gradients has been followed by time-lapse microscopy and quantified by cell tracking based on image analysis techniques. The results are expressed in terms of chemotactic index (I) and average cell velocity. The assay has been tested by comparing the migration of human neutrophils in isotropic conditions and in the presence of an Interleukin-8 (IL-8) gradient. In the absence of IL-8 stimulation, 80% of the cells showed a velocity ranging from 0 to 1 µm/min. However, in the presence of an IL-8 gradient, 60% of the cells showed an increase in velocity reaching values between 2 and 7 µm/min. Furthermore, after IL-8 addition, I increased from 0 to 0.25 and 0.25 to 0.5, respectively, for the two donors examined. These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix. The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.

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Related in: MedlinePlus

Fraction of motile cells as a function of the time for neutrophils A) from donor A, B) from donor B.The vertical line indicates the moment when the IL-8 solution was added to the chemoattractant reservoir.
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pone-0052251-g005: Fraction of motile cells as a function of the time for neutrophils A) from donor A, B) from donor B.The vertical line indicates the moment when the IL-8 solution was added to the chemoattractant reservoir.

Mentions: A cell was considered motile in a given time interval if its total displacement exceeded its own diameter. This criterion was used to exclude values representing minor cellular displacements that might be caused by either the repositioning error of the microscope motorized stage or by external perturbations of the system. The fraction of mobile cells was calculated for a time interval of 10 minutes in presence and absence of IL-8 for donors A and B, and is reported in Figure 5 A and B, respectively; the vertical line at 30 minutes from the start of the experiment shows the time IL-8 solution was added. A different percentage of motile cells was clearly visible in unstimulated conditions (Pre IL-8) for the two samples with donor B showing a higher motility. However, in both donors the stimulation of the cells by IL-8 induced a significant increase in the number of motile cells as detected in the Post time stages. The evolution over time of the fraction of motile cells suggests that at short times cells are not affected by the presence of chemoattractant, but the motile fraction progressively increases, until a maximum is reached which is then followed by a decrease at later times when the influence of the chemoattractant starts to vanish.


A novel chemotaxis assay in 3-D collagen gels by time-lapse microscopy.

Vasaturo A, Caserta S, Russo I, Preziosi V, Ciacci C, Guido S - PLoS ONE (2012)

Fraction of motile cells as a function of the time for neutrophils A) from donor A, B) from donor B.The vertical line indicates the moment when the IL-8 solution was added to the chemoattractant reservoir.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526591&req=5

pone-0052251-g005: Fraction of motile cells as a function of the time for neutrophils A) from donor A, B) from donor B.The vertical line indicates the moment when the IL-8 solution was added to the chemoattractant reservoir.
Mentions: A cell was considered motile in a given time interval if its total displacement exceeded its own diameter. This criterion was used to exclude values representing minor cellular displacements that might be caused by either the repositioning error of the microscope motorized stage or by external perturbations of the system. The fraction of mobile cells was calculated for a time interval of 10 minutes in presence and absence of IL-8 for donors A and B, and is reported in Figure 5 A and B, respectively; the vertical line at 30 minutes from the start of the experiment shows the time IL-8 solution was added. A different percentage of motile cells was clearly visible in unstimulated conditions (Pre IL-8) for the two samples with donor B showing a higher motility. However, in both donors the stimulation of the cells by IL-8 induced a significant increase in the number of motile cells as detected in the Post time stages. The evolution over time of the fraction of motile cells suggests that at short times cells are not affected by the presence of chemoattractant, but the motile fraction progressively increases, until a maximum is reached which is then followed by a decrease at later times when the influence of the chemoattractant starts to vanish.

Bottom Line: The results are expressed in terms of chemotactic index (I) and average cell velocity.These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix.The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Ingegneria Chimica Università di Napoli Federico II, Naples, Italy.

ABSTRACT
The directional cell response to chemical gradients, referred to as chemotaxis, plays an important role in physiological and pathological processes including development, immune response and tumor cell invasion. Despite such implications, chemotaxis remains a challenging process to study under physiologically-relevant conditions in-vitro, mainly due to difficulties in generating a well characterized and sustained gradient in substrata mimicking the in-vivo environment while allowing dynamic cell imaging. Here, we describe a novel chemotaxis assay in 3D collagen gels, based on a reusable direct-viewing chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane. The diffusion process has been analysed by monitoring the concentration of FITC-labelled dextran through epifluorescence microscopy and by comparing experimental data with theoretical and numerical predictions based on Fick's law. Cell migration towards chemoattractant gradients has been followed by time-lapse microscopy and quantified by cell tracking based on image analysis techniques. The results are expressed in terms of chemotactic index (I) and average cell velocity. The assay has been tested by comparing the migration of human neutrophils in isotropic conditions and in the presence of an Interleukin-8 (IL-8) gradient. In the absence of IL-8 stimulation, 80% of the cells showed a velocity ranging from 0 to 1 µm/min. However, in the presence of an IL-8 gradient, 60% of the cells showed an increase in velocity reaching values between 2 and 7 µm/min. Furthermore, after IL-8 addition, I increased from 0 to 0.25 and 0.25 to 0.5, respectively, for the two donors examined. These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix. The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants.

Show MeSH
Related in: MedlinePlus