Hq2 cooled ccd camera

Live cell imaging was performed on a temperature-controlled inverted Nikon Ti-E microscope with a Lumen 200 Pro light source (Prior) and an HQ2 cooled CCD camera (Roper Scientific) controlled via Metamorph acquisition software (MDS Analytical Technologies). Live cell movies of transfected cells were collected on an inverted Nikon Ti-E microscope with a CSU-X confocal scanhead (Yokogawa), laser merge module containing a 491 laser line (Spectral Applied Research) and an HQ2 cooled CCD camera (Roper Scientific). Motility and protrusion data was obtained using either a 10X or 20x objective (Nikon); Immunofluorescence Images were obtained using a 40x 1.3 NA Plan Fluor objective (Nikon).

Live cell traction force measurements were performed on an inverted Nikon Ti-E microscope with a CSU-X confocal scanhead (Yokogawa), laser merge module containing 491, 561 and 642 nm laser lines (Spectral Applied Research) and an HQ2 cooled CCD camera (Roper Scientific). All hardware was controlled via Metamorph acquisition software (MDS Analytical Technologies). Traction force data was obtained at 37°C in a perfusion chamber (Warner Instruments) using a 60x 1.2 NA Plan Apo WI objective (Nikon). Cells were maintained in culture media supplemented with 10 mM HEPES and 30 μl/ml Oxyrase (Oxyrase, Inc.).

Micropipette aspirations were visualized on an inverted Nikon Ti-E microscope with with a Lumen 200 Pro light source (Prior) and an HQ2 cooled CCD camera (Roper Scientific). The observation chamber is made of two glass coverslips separated by a Parafilm spacer. A glass micropipette, pulled from borosilicate capillaries (0.7/1.0 mm inner/outer diameter, Kimble, Vineland, NJ) with a laser puller (Sutter P2000) and microforged (DMF1000, World Precision Instruments, Aston, UK) to a diameter of 4–6 µm, is inserted into the chamber. The chamber is filled with a 0.5 mg/mL casein solution for 30 minutes to passivate all glass surfaces. Then the casein solution is replaced by cell culture medium. The aspiration pressure is adjusted by controlling the height of a mobile water tank connected to the micropipette. A cell is selected and the micropipette is brought close to the cell membrane with a 0 aspiration pressure. Then the aspiration pressure is increased from 0 Pa to 588 Pa while images of the cell are recorded. The length of the cell portion aspirated inside the micropipette is then measured with ImageJ.