Cool snapfx

Cells were grown on collagen‐coated glass coverslips (35 mm, Ibidi GmbH) for 48 hours then stimulated for 20 minutes with HGF (0.5 nM) and observed with an inverted microscope Axiovert 200 M (Carl Zeiss, Inc.), and ×100 plan Apochromat NA 1.4 objective, AxioCam HRM (Carl Zeiss, Inc.). Thirty‐one images per cell were acquired at 4 seconds intervals using a digital camera (charge‐coupled device Coolsnap FX (Princeton Instruments); Coolsnap HQ (Roper Scientific). Plus‐ends of individual MTs were tracked in time using MetaMorph software (MDS Analytical Technologies). The number of catastrophes (transition from growth or pause to shortening) and rescues (transitions from shortening to pause or growth) was calculated as described previously40 from 30 MTs in three independent experiments. Means and (±) SE of the mean (SEM) are shown.

Isolated cells were preloaded with a standard external solution containing 2 μM Fura-2/AM (Invitrogen, Oregon, USA) for 40 min at 37°C. Ca²⁺ images were captured using an inverted fluorescence microscope (Olympus IX70, Japan) and a silicon intensifier target camera (Cool SNAPfx, Roper Scientific), and recorded on a fluorescence-imaging system (LAMBAD 10-2, Sutter Instrument Co, USA). For measurement of [Ca²⁺]_i with Fura-2, the excitation wavelengths were 340 nm and 380 nm, a dichroic mirror of 440 nm and an emission filter of 510 nm were used. [Ca²⁺]_i was calculated from the ratio (R) of the two fluorescence intensities, F340/F380 [22 (link)]. The change in [Ca²⁺]_i was calculated by subtracting [Ca²⁺]_i at the peak response level from that at the pre-stimulated level.

Cells grown on glass coverslips were washed with PBS, fixed with 4% paraformaldehyde or with methanol for gp74 staining, blocked, and permeabilized with PBTG buffer (0.1% Triton X-100, 1% bovine serum albumin [BSA], and 1M glycine in PBS) for 15 min at room temperature. Samples were incubated with primary antibodies diluted in 1% BSA in PBS for 1 h at room temperature, washed with PBS, and incubated with fluorescently labeled secondary antibodies for 30 min at room temperature, as described (García-Briones et al., 2006 (link)). After washing with PBS, nuclei were stained with 1 μg of DAPI (4′,6′-diamidino-2-phenylindole; Invitrogen)/ml or with To-Pro-3 (Molecular Probes). Samples were mounted in Fluoromount G (Southern Biotech) and observed with an Axioskop microscope (Zeiss) coupled to a digital monochrome camera coolsnap FX (Roper Scientific). Images were acquired by using RS Image 1.9.2 software (Roper Scientific) and processed using Adobe Photoshop CS2 (Adobe Systems, Inc.). For confocal laser scanning microscopy, samples were observed using a Leica TCS SPE confocal lases scanning microscope. Images were acquired using Leica Advanced Fluorescence Software and processed with Adobe Photoshop CS2 (Adobe Systems, Inc.). Optical slice thickness for all confocal images displayed was of 1 airy unit.

Cells were subjected to apoptotic stress by incubating with camptothecin (CPT) (20 μM, 37 °C, 0–8.5 h) as described previously24 (link). Hoechst 33258 (10 ng/ml) was added to the culture medium for 10 min at 37 °C. All images were collected using a Nikon TE2000 fluorescence inverted microscope equipped with a CoolSnap FX charge-coupled device camera (Roper Scientific, Trenton, NJ, USA) and MetaMorph image analysis software 4.01 (Universal Imaging, Dowington, PA, USA). Apoptotic cells were quantified using Guava easyCyte by flow cytometer as described before25 (link). Confocal images were obtained using the Olympus Fluoview FV1200 Laser scanning confocal Microscope using a 60X NA1.35 objective.