Volocity software 6

An A1R MP+ microscope (Ti Microscope) equipped with a Plan Apo λ 10X objective from Nikon Instruments was used for time lapse studies of dyscohesion and migration. Brightfield live-cell imaging was performed with a temperature-controlled heated stage at 37 °C over ~48 h and images were taken every 25 min. Images were imported into Volocity Software 6.0.1 (PerkinElmer Inc., Waltham, MA, USA) to calculate the area of cell dispersion from microtumors at the indicated time points. To represent the total microtumor dispersion area, the drawn perimeter consisted of spreading cells migrating out of the microtumors and free the spaces created by migratory cells at later time points. Briefly, regions of interest at the perimeter of spreading cells were defined using the Free Hand tool at the microtumor periphery (yellow dotted lines in Figure 5) during the initial time points; and as cell dispersion from microtumors occurred. The few cells that left microtumors as single cells were not included in the analysis. The area values (µm²) were exported in a comma separated value (.csv) format. Using Excel, the change fold in area was calculated by dividing spreading area at the indicated time points by the spreading area at time 0. Change fold in area values were analyzed using GraphPad Prism 9.0.1 software (San Diego, CA, USA).

10μl of antibody stained bacteria preparation was pippetted onto a 76x22mm glass slide (Menzel Gl00E4ser, Germany) and a 22x22mm glass cover slip (Menzel Gläser, Germany) placed over the sample. Samples were heated for 60 minutes at 50°C, and observed at X40 and X100 magnification using a Zeiss Axioscope M1 fluorescence microscope with the correct filters. Images were analysed in Volocity software 6.0.1 (Perkin Elmer). Images were arranged in Adobe Photoshop elements 10.

BAL was performed to obtain leukocytes present in the alveolar space. Mice were killed by anesthetic overdose and the trachea of each animal was exposed and cannulated with a polypropylene catheter of 1.7 mm. Airways were washed with 2 ml of ice-cold PBS. Total cell counts were performed in a modified Neubauer chamber using Turk's stain. Differential cell counts were performed on cytocentrifuge preparations (Shandon Cytospin III), stained with May–Grünwald–Giemsa using standard morphological criteria to identify cell types. The results are presented as the number of cells/BAL. In a separated set of experiments, apoptotic cells were morphologically identified in cytocentrifuged slides, which were also positively stained for cleaved caspase-3 (Alexa Fluor 488 rabbit anti-mouse cleaved caspase-3; Cell Signaling; 1 : 50). Fluorescence intensity was measured offline using Volocity software 6.3 (Perkin–Elmer, Waltham, MA, USA) and fluoresce profile was assessed using Image J (NIH).^{57 (link)}

DSBs were generated in 0.25 × 3 µM tracks using a Stanford Research Systems (SRS) NL100 nitrogen MicroPoint System (Andor Technology, Belfast, United Kingdom) equipped to a Nikon Eclipse TE2000 spinning disk confocal microscope (Nikon Instruments, Melville, NY, USA). The microscope was supported with temperature and CO₂‐regulated incubation chamber, and the DSBs were induced with 435 nm laser regulated through Volocity software 6.3 (PerkinElmer, Waltham, MA, USA). Following the laser‐induced DSBs, mCherrry‐WRN recruitment was recorded at 5 to 15 sec intervals for 5 min with a CCD camera (Hamamatsu, Hamamatsu City, Shizuoka, Japan). The fluorescence intensity of the damaged area was measured with Volocity imaging software and normalized to that of a control area. The results are presented as mean ± SEM, and p‐values were measured with Student's t test.

Generation of laser-induced DSB and recruitment of fluorescence protein-tagged proteins were performed with a SRS NL100 nitrogen MicroPoint system equipped to a Nikon Eclipse TE2000 spinning disk confocal microscope with Volocity software 6.3 (Perkin-Elmer)^{34 (link),64 (link)}. For treatment of CDK inhibitors, U2OS cells expressing GFP/YFP-tagged RECQL4 and YFP-MRE11 were pre-incubated with DMSO, 20 µM Roscovitine (Santa Cruz), 10 µM CDK1 inhibitor RO3306, 10 µM CDK2 inhibitor 2-III (EMD Millipore) for 4 h under standard cell culture conditions, and then subjected to micro point laser-irradiation. To study the function of CDK-dependent phosphorylation of Ser89 and Ser251 on RECQL4 recruitment to DSBs, GFP-tagged WT RECQL4, RQ4-2A, and RQ4-2D were expressed by transfecting the plasmids pEGFP-C1-RQ4Wt, pEGFP-C1-RQ4-2A, and pEGFP-C1-RQ4-2D into U2OS cells, respectively. For CDK 1and CDK2 inhibition, the U2OS cells were incubated with RO3306 and CDK2 inhibitor-III for 4 h before laser irradiation. The recruitment of these proteins was conducted as described above. The fluorescence intensity of the damaged area was measured with Volocity imaging software and normalized to that of a control area. The results are presented as mean ± s.e.m., and P -values were measured with Student’s t-test.

Liver microcirculation was imaged as described previously [29 (link), 40 (link)]. Briefly, mice were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (15 mg/kg). The liver was gently pulled out through a laparotomy incision and positioned over a Plexiglas stage for imaging under a confocal inverted microscope (Nikon Eclipse Ti and C2 confocal head). For imaging liver microcirculation and necrosis, mice received endovenously 8 μg of phycoerythrin-labeled anti-PECAM-1 (stains liver sinusoidal endothelial cells; eBiosciences, USA) and 2 μL of the stock solution of Sytox Green (stains extracellular DNA deposits and necrosis; Life Technologies, USA). The liver surface was imaged in a custom made stage that holds the liver in flat position. Fluorophores were excited with 488 and 543 nm lasers line using a 10x objective. All images were generated by Volocity software (6.3; PerkinElmer, USA).

At Day 10 after the transplant, mice were killed and lymphoid organs (lymph node and spleen) prepared for confocal microscopy analysis. Images were obtained using C2 Eclipse Ti confocal microscope (Nikon). Total cell counts were performed in a modified Neubauer chamber using Tripan’s stain. Tumor cells (GFP+P815 cell) were identified in cytocentrifuged slides, which were also stained with DAPI to nuclear stain. Fluorescence intensity was measured off-line using Volocity software 6.3 (Perkin-Elmer) and the fluorescence profile was assessed using Image J (NIH).