Orca er camera

Live cell AFM experiments were carried out as previously described^{6 (link)}. AFM experiments were carried out in a Nanowizard 4 AFM (JPK) mounted on top of a Nikon Ti Eclipse microscope, using the JPK software (JPK Data Processing Version 6.1.79). Polystyrene beads of 20 μm were attached using a non-fluorescent adhesive (NOA63, Norland Products) to the end of tipless MLCT cantilevers (Veeco). The spring constant of the cantilevers was calibrated by thermal tuning using the simple harmonic oscillator model. Experiments were carried out on cells previously transfected with the different constructs indicated in figures, incubated with Hoechst 33342 (Invitrogen), and seeded on 1.5 kPa gels. For each cell, the nucleus was identified by using the Hoechst fluorescence signal, and a force of 1.5 nN was applied to the nucleus. Once the maximum force was reached, the indentation was kept constant under force control, adjusting the z height by feedback control. An image was acquired every 10s by an Orca ER camera (Hamamatsu) and a 60X (NA = 1.2) objective.

For immunofluorescence, cells were seeded on glass coverslips in 35-mm culture dishes. For double staining for E-cadherin and β-actin or zyxin, cells were fixed with 1% paraformaldehyde (PFA) at room temperature for 10 min and permeabilized with cold methanol for 3 min. For double staining for actin and p34 or β-catenin, or triple staining for β-catenin/actin/EPLIN, cells were fixed with 3.7% PFA at room temperature for 15 min and permeabilized with 0.5% Triton X-100 for 5 min. Fixed specimens were incubated for 40 min with primary antibodies and subsequently, for 40 min with secondary antibodies. Mounted samples were examined with a Leica TCS SP5 confocal laser scanning microscope equipped with an HDX PL APO 63× objective or with a Nikon Eclipse Ti-E microscope equipped with a Plan Fluor 40× objective and ORCA-ER camera (Hamamatsu Photonics) controlled via NIS-Elements AR 3.22 software (Nikon).

For time-lapse microscopy, cells were seeded in 96-well μClear plates (Greiner) at 1.3 × 10⁴ cells per well in a volume of 100 μl. Synchronization was performed by adding thymidine at a final concentration of 2 mM four hours post-seeding then removed 16 hours later by washing three times with PBS followed by adding fresh media. Four hours after thymidine washout, additional media containing inhibitor(s) was added and imaging then started one hour later. Fluorescence imaging was then performed using a Pathway Bioimager 855 (BD Biosciences), BD Attovision software and a 20x/0.30 UPlan FLN objective. 96-well plates were housed in a customized chamber to maintain a constant temperature of 37°C and a humidified 5% CO₂ atmosphere. Images were collected every 5 min as a 2 × 2 montage using an Orca ER camera (Hamamatsu) with a 0.1 s exposure time. For analysis of daughter pairs, imaging was performed for 24 hours in drug-free media prior to addition of fresh media containing inhibitor. Image sequences were then viewed using NIH ImageJ software. Cells were tracked manually and behavior determined by visual inspection of GFP histone H2B morphology. Timing data was analyzed and sorted using Microsoft Excel then cell fate profiles and other graphs created using GraphPad Prism.

HeLa Kyoto cells were fixed with 4% formaldehyde at room temperature (RT) for 20 min or in methanol at −20 °C overnight and processed for immunofluorescence microscopy (IF) as described45 (link)46 (link). For 53BP1 staining, cells were treated with pre-extraction buffer (10 mM Pipes, pH 7, 100 mM NaCl, 300 mM sucrose, 1.5 mM MgCl₂, 5 mM EDTA and 0.5% Triton X-100) for 5 min on ice, then fixed with 4% formaldehyde for 20 min at RT. Primary antibodies used are listed in Supplementary Table 1. Alexa fluorophore-conjugated secondary antibodies were used for detection. DNA was stained using DAPI. Images were acquired using an Olympus FV1000D (InvertedMicroscopeIX81) confocal laser scanning microscope equipped with a PlanApoN × 60/1.40 NA Oil Sc objective lens controlled by FV10-ASW software. For visual inspection and scoring a Zeiss Axio Imager M1 microscope using Plan Neofluor × 40/1.3 oil objective lens (Zeiss) equipped with an ORCA-ER camera (Hamamatsu) and controlled by Volocity 5.5.1 software (Improvision) was used.