Axiocam hrm ccd camera

EA.hy926 cells were seeded in 0.2% gelatin-covered μ-Slide I^0.4 at 0.5 × 10⁵ cells per slide and incubated in EGM-2mv growth medium for 1.5 days until subconfluence. Growth medium was changed every 12 hours. Cells were washed with HBSS and challenged with 250 μM dicarbonyl solution in HBSS for 4 hours in a CO₂ incubator. Then, the μ-Slide was placed under the microscope without washing out dicarbonyls. Time-lapse recording of live EA.hy926 cells was performed using a motorized Axiovert 200M inverted microscope equipped with an on-stage thermostat (37°C) and an AxioCam HR_m CCD camera (Zeiss, Germany). Movies were recorded using a 40x objective in the phase-contrast mode taking one frame per 20 sec for 1 hour. Images were collected and processed using the AxioVision 4.8.2 software (Zeiss, Germany), the ImageJ freeware (NIH, Bethesda, USA), and Adobe Photoshop CS6 (Adobe Corp., USA).
Lamellipodial activity of individual cells was quantified from these movies using the ImageJ freeware. The perimeter of a lamellipodium that expanded/retracted from its initial position during 100 sec period was traced using a freehand selection tool. It was then converted in the number of pixels contained within the outlined perimeter using the “measure” function of ImageJ. Measurements from 10–12 cells were averaged and expressed as the speed of lamellipodial area expansion/retraction in pixels/min.

After being grown and transfected as described, the cells were fixed for 20 min in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and permeabilised with 0.5% Triton X-100. Immunostaining with primary antibodies was followed by incubation with Fluorescein isothiocyanate (FITC)/CY5 anti-rabbit/mouse antibodies (Jackson ImmunoResearch). Texas-red Phalloidin was used to stain F-actin (Molecular Probes). Wide-field microscopy was performed with a Zeiss Axioplan inverted phase-contrast microscope (60x objective) connected with an AxioCam HRm CCD camera.

Wild type mid-log phase L. amazonensis promastigotes were exposed to different starvation conditions for 24 h. Cells were then washed, fixed and coverslips were mounted. Phase contrast images of parasites were captured at 100x magnification with a Zeiss Axiovert 200M fluorescence microscope equipped with a AxioCam HRm CCD camera.
Motility patterns of the parasites were examined by video-microscopy. Mid-log phase parasites were grown in either complete medium or specific nutrient-depleted medium for 24 h. Cells were then observed using the 40x objective of an Olympus IX73 inverted microscope. Videos were captured with a QImaging Retiga 6000 monochrome CCD camera.

Cells adherent to Lab-Tek chamber slides (177,380) were rinsed in PBS. The cells were treated with BRB80 buffer for 5 min and then fixed for 10 min with 3.7% Formaldehyde in BRB80 buffer. After washing the cells in PBS for 5 min, they were treated with PBST (PBS+0.1% Triton X-100) for 15 min. Finally, the cells were blocked for 20 min in PBST containing 3% BSA. Both control and Tum/RacGAP dsRNA-treated cells were stained with anti-Tubulin diluted 1:200 (S9026 Sigma T clone DM1α), anti-Pav/kinesin-6 diluted 1:200 (kindly provided by D. Glover) or anti-Feo diluted 1:100 (Feo, kindly provided by M. Gatti) antibodies. Immunostained preparations were analysed with a Zeiss Axioskop 2 plus fluorescent microscope equipped with an AxioCam HRm CCD camera and images were acquired with a Plan-NEOFLUAR × 100 1.30 oil objective and Axiovision 4.6.3 software (Zeiss). Photoshop CC and Illustrator CC (Adobe) software were used to prepare the images. To confirm that the Pav/kinesin-6 signal observed in control and Tum/RacGAP RNAi-treated cells was specific to Pav/kinesin-6 primary antibody and not the result of non-specific binding or auto-fluorescence of the secondary antibody, we stained the cells with the Alexa Fluor 594 goat anti-rabbit secondary antibody alone.

Cells were washed with PBS (Sigma) and fixed in 4% paraformaldehyde for 30 min at room temperature. Then, cells were permeabilized in CSK buffer (10 mM HEPES pH 7.4, 300 mM Sucrose, 100 mM NaCl, 3 mM MgCl₂) with 0.1% Triton-X for 30 min. Blocking was performed for 1 h with 5% FBS in PBS after which cells were stained in 1% FBS in PBS overnight at 4 °C. After primary staining, cells were washed three times 5 min with PBS and incubated with secondary antibodies for 1 h at RT. After another three 5 min washes in PBS, slides were mounted with Aqua-Poly/Mount (Polysciences). Images were obtained using an AxioObserver Z1 confocal spinning-disk microscope (Zeiss) equipped with an AxioCam HRm CCD camera (Zeiss) or a sCMOS ORCA Flash 4.0 camera (Hamamatsu) and a Plan/Apo 63 Å~/1.4 water‐immersion objective. The percentage of cells with nucleolar retention of γH2AX foci were defined as cells that show an overlap (yellow) of the γH2AX (green) with the NPM (red) signal. Blinded visual scoring of rDNA damage retention was performed by two individuals. Graphs were generated using Graphpad Prism and Inkscape.

PriGO8A cells transduced with doxycycline inducible Lgl3SA were plated on laminin coated Bioptechs delta-T dishes (Butler, PA, USA) in 1mL media. Cells were grown for three days at 37°C in 5% O₂/CO₂ in the presence or absence of 500 ng/mL doxycycline in the media. Cells were maintained at 37°C in a sealed chamber for the duration of the image acquisition. Phase contrast images of the cells were taken at 5 min intervals for 17 h using the through 10x objective of the ZiessAxiovert 200 M microscope equipped with a AxioCamHRm CCD camera(Zeiss, Göttingen, Germany). Motility was quantified as the average distance per point every five frames of ten cells per condition using the MtrackJ plugin [26 (link)] in ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

Slides were examined using the Axioimager D1 microscope (Carl Zeiss, Jena, Germany) equipped with the Axiocam HRm CCD camera (Carl Zeiss, Jena, Germany), Carl Zeiss filter sets (FS01, FS38HE, and FS43HE), and the image-processing AxioVision Release 4.6.3. software (Carl Zeiss, Jena, Germany). All preparations were mounted with the Vectashield antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA).