Orca 2 camera

Dynamics of CDR formation was imaged by differential interference contrast (DIC) on an Olympus VivaView FL microscope using a UPLSAPO 20×, numerical aperture 0.75, DIC objective and a Hamamatsu ORCA II camera. Cells were stabilized for 30 min in the incubator before the addition of PDGF. Images were acquired every 35 s. Six different samples (three CTRL and three RhoG KD) were analyzed in parallel for every experiment, and five fields were imaged simultaneously for each of the samples. CDR properties were measured in every frame for each ruffle using ImageJ as described.

Chromosome spreads were performed as previously described (Wahba et al., 2013 (link)). Slides were incubated with a mouse anti-FLAG monoclonal (Sigma-Aldrich) at 1:2000, mouse anti-V5 (Life Technologies, Carlsbad, CA) at 1:2000, polyclonal rabbit anti-Pds5 at 1:2000, or polyclonal rabbit anti-Mcd1 at 1:2000 dilutions. The primary antibody was diluted in blocking buffer (5% bovine serum albumin, 0.2% milk, 1× phosphate-buffered saline, and 0.2% Triton X-100). The secondary Cy3-conjugated goat anti-mouse antibody (115-165-003) was obtained from Jackson ImmunoResearch (West Grove, PA) and diluted 1:2000 in blocking buffer. Indirect immunofluorescence was observed using an Olympus IX-70 microscope with a 100×/NA 1.4 objective and Orca II camera (Hamamatsu, Bridgewater, NJ).

Cells grown on 22-mm² coverslips were simultaneously fixed and permeablized with 2% paraformaldehyde and 0.5% Triton X-100 in 1X PHEM buffer at room temperature for 15 min. The cells were blocked with 20% boiled normal goat serum (BNGS) for at least 20 min. Coverslips were incubated with primary antibody, Anti-­centromere antibody (1:800, Antibody Inc., 15-134), and rabbit anti SGO1 (1:500, a gift from Hongtao Yu, University of Texas Southwestern Medical Center) diluted in 5% BNGS in PBST overnight at 4°C. Coverslips were washed three times with MOPS buffered saline with 0.05% Tween 20 and then incubated in secondary antibody, goat anti-rabbit conjugated to CY3 at 1:1500 (JacksonImmuno­Research, 111-165-045109), and goat anti-human conjugated to fluorescein isothiocyanate at 1:800 (JacksonImmunoResearch, 109-95-088) for 2 h at room temperature. After incubation with secondary antibodies, coverslips were washed three times again and then labeled with DAPI (100 ng/ml) for 1 min. Coverslips were mounted on slides with Vectashield mounting media (Vector Laboratories, H-1000) and then sealed with clear nail polish. Fluorescence images of cells were taken using a Zeiss Axioplan II microscope with a Zeiss 100× objective, Hamamatsu Orca II camera, and Metamorph software. Distances between pairs of kinetochores were measured using the region measurement tool in Metamorph software.