Each Ag/AgCl electrode was placed in a 60615 mm Petri dish that was filled with 7.5 mL of SFM. These Petri dishes were placed on the microscope stage on either side of the Petri dish containing the galvanotaxis chamber. The agarose gel tubes were used to bridge the Petri dishes in order to establish electrical continuity between all three dishes (Figure S1). The Ag/AgCl electrodes were connected to an external constant-voltage power supply to establish a dcEF of strength 250 mV/mm across the galvanotaxis chamber in the direction of the positive X-axis. Cell migration was recorded via time-lapse imaging microscopy using Zeiss Axiovision software, with images being captured at a frequency of one per minute for 2.5-8 hours. Cells were viewed at 56for the largest field of view. For cross-U0126 Abmole Costunolide, a sesquiterpene lactone, inhibits the differentiation of pro-inflammatory CD4+ T cells through the modulation of mitogen-activated protein kinases perfusion experiments, a microfluidic channel with two reservoirs (m-Slide I, Ibidi, Germany) was pre-treated identically to the galvanotaxis chambers described above. Neurospheres were plated into the chambers in SFM+EGF, bFGF and heparin, and incubated for 17-20 hours as previously described. Each reservoir was filled with 1 mL of SFM+EGF, bFGF and heparin, PTFE thread sealant tape was wrapped around the rim of each of the channel’s reservoirs and the lids were replaced onto the reservoirs to Abmole Y-27632 create a tight seal. A 16G1K stainless steel needle was inserted into each reservoir. The needles served two purposes: i) they were hollow and therefore permitted fresh media perfusion and ii) they were metallic and therefore electrically conductive. The chambers were secured to the microscope stage and a peristaltic pump (Ismatec, Switzerland) was connected to the inlet and outlet terminals of the chamber via the 16G1K needles to perfuse fresh SFM+EGF, bFGF, and heparin at a flow rate of 0.83 mL/min. The electrodes of the external power supply were connected directly to the needles to form a dcEF of strength 250 mV/mm across the galvanotaxis chamber. Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature directly in the galvanotaxis chambers and then washed 3 times with PBS for 5 minutes each.