Chemotaxis in μ-Channels

Inhibitor of NF-κB Kinases α and β Are Both Essential for High Mobility Group Box 1-Mediated Chemotaxis

“…Importantly, WT versus IKKβ −/− or WT versus IKKα −/− MEFs (generated from WT cells by 4-OHT–induced IKK deletions) were directly compared in the same HMGB1 gradient by distinguishing them with different fluorescent tags. Analogous versions of this technique have been extensively used to tease out fine details of cellular movement in response to cytokine and chemokine gradients in physiologically well-controlled environments (51, 55–57). …”

“…After cells had firmly attached to the substratum, slides were laid on a 37°C humidified stage of an UltraVIEW European Remote Sensing spinning disk confocal microscope (Perkin Elmer, Waltham, MA). A 0–30 μg/ml HMGB1 gradient, mixed with fluorescent beads (Invitrogen, San Diego, CA) for microscope viewing, was allowed to form in the chamber’s channel as described (51) (Supplemental Fig. 4).…”

Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling

“…To begin to explore the mechanism of action of IKKα in CXCL12 induced cell migration, we dissected the migration parameters of WT vs. induced IKKα KO primary MEFs in time-lapse microscopy experiments employing IBIDI cell migration slides (30, 43). In vitro migration assays were performed with primary IKK α f/f ;CreERT2 MEFs sham-treated (WT control) or treated with 4-OHT (IKKα induced deletion) in IBIDI μ slides as previously described (30).…”

“…The functional impact of IKKα for directional chemotaxis is best determined at the earliest phase of the migration response, which defines cellular polarization/orientation towards the CXCL12 gradient. In this analysis, we defined directional tracks as those with ending points closer to the higher CXCL12 concentration in the gradient, compared to their starting points, whilst non-directional tracks are the opposite; and indeterminate tracks starting and ending at the same distance from the CXCL12 gradient (moving laterally) are also considered non-directional tracks {see detailed description of how the IBIDI experiments were performed in Materials and Methods} (30, 43). These time lapse time video microscopy experiments revealed that IKKα is critical for determining initial cellular orientation/polarization towards a CXCL12 gradient (compare directions of migration tracks in WT vs, induced IKKα KO cells in Figure 4B and 4C respectively and see statistically analyzed bar graph results summary in Figure 4C).…”

“…After cells had firmly attached to the substratum, slides were laid on a 37°C humidified stage of an UltraVIEW ERS Spinning Disk confocal microscope (Perkin Elmer). A 0-30 μg/ml CXCL12 gradient, mixed with fluorescent beads (Molecular Probes) for microscope viewing, was allowed to form in the chamber's channel as described (43). Pictures of cells at the edge of the gradient were captured every 2 minutes for up to 3 h (microscope objective Zeiss, 5X magnification, numerical aperture 0.15; EM-CCD Hamamatsu C9100 Camera; UltraVIEW ERS acquisition software).…”