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At all phases of migration, cells were seen to migrate tangentially in one direction, only to reverse direction at a later time.
To assess transwell migration, cells were labeled with 5 μg/ml of the green fluorescent tracer DiO (Life Technologies).
After 3 h of migration, cells were fixed (4% formaldehyde in PBS) and permeabilized (0.1% Triton X-100 in PBS).
To study the contribution of MTs to T cell polarization and migration, cells were treated with MT stabilizing (taxol) or depolymerizing (nocodazole) reagents.
After 16 h of incubation, the migration cells were fixed and stained using crystal violet staining.
For random migration, cells were seeded on Matrigel-coated 24-well plates 48 h after transfection.
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Four scratched fields were randomly chosen and the number of migration cells was counted.
The number of migration cells was counted under a microscope for five random areas at 200× magnification.
For migration assays, cells were seeded onto Transwell with 8.0 μm Pore Polycarbonate Membrane Inserts (Corning).
To calculate cell migration velocity, cells were tracked using the timelapse macro in Axiovision software.
Cell migration assay: Cells were transfected with miRNAs and controls as described (Spaderna et al, 2006).
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