Eclipse te 2000 4 microscope

Cells were treated with control and BPAG1 siRNA for 24 h as above and cultured in a 12-well dish. Either one scratch per well or 3 parallel scratches and 1 scratch perpendicular to them to make 3 intersections (crosses) were applied to the cell monolayers using a 200 µl pipette tip. The medium was immediately changed to fresh proliferation medium to remove the detached cells. Then, a phase contrast picture was taken of each scratch cross immediately and 19 h later using Eclipse TE-2000-4 microscope (Nikon). Wound areas were measured using ImageJ. For time lapse microscopy, cells with one scratch per well were switched to fresh proliferation medium with 25 mM HEPES and placed under the Eclipse TE-2000-4 microscope equipped with a 37°C chamber, recording an image of the scratch every 10 min for 19 h. Velocity and directness of cell migration were measured by manual tracking and chemotaxis and migration plugin (Ibidi) in ImageJ.

Approximately 1 × 10⁶ cells were loaded with the calcium-binding dye, Fluo-3 AM (3.5 μM in DMSO with 0.01% pluronic acid) and incubated in the dark for 60 min at room temperature. Cells were viewed using an inverted Nikon Eclipse TE2000-4 microscope (Mississauga, ON, Canada) with a 20× objective. Intracellular calcium recordings were obtained using a custom-built apparatus previously described by Mukherjee et al. (24 (link)). During recordings, eosinophils were scanned with a 20 mW photodiode laser (Coherent Technologies, CA, USA) at 488 nm in the X- and Y-planes using two mirrors oscillating at 8 and 30 Hz, respectively. The fluorescence light emitted at 510 nm was measured by a photomultiplier. Video Savant v4.0 software (IO Industries, London, ON, Canada) was used to digitize the signal (8-bit resolution) and produce tiff images (480 × 400 pixels) at a rate of 30 Hz. Videos were created by recording filtered frames (obtained by averaging eight consecutive raw images). Unless otherwise noted, all recordings were obtained at a frame rate of 2 Hz.