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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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Cell trajectories projected on the XY plane and referred to the same origin.A: Random motion in absence of any chemical stimulus. B: In the presence of an IL 8 concentration gradient (C0 = 50 µg/ml) a preferential direction is qualitatively evident: the cells migrate towards the negative Y direction, i.e. in the direction of the membrane, towards the chemoattractant gradient.
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pone-0052251-g003: Cell trajectories projected on the XY plane and referred to the same origin.A: Random motion in absence of any chemical stimulus. B: In the presence of an IL 8 concentration gradient (C0 = 50 µg/ml) a preferential direction is qualitatively evident: the cells migrate towards the negative Y direction, i.e. in the direction of the membrane, towards the chemoattractant gradient.

Mentions: Figure 3.A shows the trajectories of 54 cells, tracked for 110 minutes, labelled with symbols having different shapes and grey intensities, in isotropic conditions. Each trajectory is referred to the same initial position that coincides with the origin of the coordinate system. In this control experiment where no chemoattractant was added, cell trajectories were uniformly distributed in space (see Figure 3A), thus showing that cells were moving in a random orientation (i.e., no preferential direction of motion can be distinguished). Such random pattern confirms the absence of any matrix-mediated contact guidance effect. In the presence of the IL-8 concentration gradient, cell trajectories were instead directed towards the chemoattractant source as shown in Figure 3.B with results from 76 cells. Most of the cell paths have a preferential orientation toward the negative y axis, which is the direction of the concentration gradient.


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)

Cell trajectories projected on the XY plane and referred to the same origin.A: Random motion in absence of any chemical stimulus. B: In the presence of an IL 8 concentration gradient (C0 = 50 µg/ml) a preferential direction is qualitatively evident: the cells migrate towards the negative Y direction, i.e. in the direction of the membrane, towards the chemoattractant gradient.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0052251-g003: Cell trajectories projected on the XY plane and referred to the same origin.A: Random motion in absence of any chemical stimulus. B: In the presence of an IL 8 concentration gradient (C0 = 50 µg/ml) a preferential direction is qualitatively evident: the cells migrate towards the negative Y direction, i.e. in the direction of the membrane, towards the chemoattractant gradient.
Mentions: Figure 3.A shows the trajectories of 54 cells, tracked for 110 minutes, labelled with symbols having different shapes and grey intensities, in isotropic conditions. Each trajectory is referred to the same initial position that coincides with the origin of the coordinate system. In this control experiment where no chemoattractant was added, cell trajectories were uniformly distributed in space (see Figure 3A), thus showing that cells were moving in a random orientation (i.e., no preferential direction of motion can be distinguished). Such random pattern confirms the absence of any matrix-mediated contact guidance effect. In the presence of the IL-8 concentration gradient, cell trajectories were instead directed towards the chemoattractant source as shown in Figure 3.B with results from 76 cells. Most of the cell paths have a preferential orientation toward the negative y axis, which is the direction of the concentration gradient.

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