Digital sight ds fi1 ccd camera

Passage 3 to 4 equine BMSCs (n = 3) were plated onto 24-well tissue culture plates (Corning Inc., Corning, NY, USA) within silicone inserts containing a defined, 500 μm cell-free gap (Ibidi®, Martinsried, Germany) for migration assays. After 6 h, media was changed to either control media, media containing β-lactose (100 mM, 200 mM) or media supplemented with recombinant equine IL-1β (5 ng/mL, 10 ng/mL) or recombinant equine TNF-α (25 ng/ml 50 ng/ml). Twenty hours later, inserts were removed and media was replaced with control media or media containing β-lactose (100 mM, 200 mM). Phase contrast images were obtained at 0, 3, 8, 12, 24 and 48 hours following insert removal, using three images/well obtained with a × 10, NA 0.25 objective on an Olympus CK2 microscope with a Nikon Digital Sight DS-Fi1 CCD camera to image the entire cell-free gap. NIH Fiji was used to define the x,y coordinates of the leading edges of cells migrating across the cell-free region. Custom software (Python Software Foundation, Wilmington, DE, USA) was designed to measure the mean linear cell-free distance for each image, which was normalized to the cell-free distance at time zero for each treatment.

Our early attempts to establish a stable hChR2 transgenic human embryonic stem cell (hES) clonal line to avoid variable transduction and expression efficiency was hampered by problems in stable transgene expression despite the presence of the transgenes hChR2 and YFP in the genomic DNA (S1 Fig and S1 Table). After differentiation of H9 cells to NPs and neurons, transgene expression was not detectable; varying neural differentiation methods. Therefore, we decided to transduce cells at the NP stage of differentiation. Neural progenitor cells (2 million) were plated on a matrigel-coated 35mm dish on day 34. Two days after plating, cells were treated with polybrene (6 μg/mL) in NP expansion medium at 37°C for 15min. Lentivirus harboring hSyn-hChR2(H134R)-eYFP-WPRE was added to each well at moi 0 or 13 and incubated at 37°C for 24 hours. The virus medium was replaced with new NP expansion media (NPEM; DMEM/F12, N2, B27, 20ng/mL FGF2) on the next day and live cell images of NPs or neurons expressing hChR2 were acquired 4–5 days after transduction using a Nikon TS 100 fluorescence inverted microscope (Nikon Instruments Inc., Melville, NY) with a Digital Sight DS Fi1 CCD camera (Nikon Instruments Inc., Melville, NY).