CometAssay® Reagent Kit for a Single Cell Gel Electrophoresis Assay Catalog # 4250-050-K was purchased from R&D systems, and the reagents were made according to the manufacturer’s instructions. The alkaline comet assay was performed on cells treated with vehicle control and bortezomib after treatment for 24 h, according to the manufacturer’s instructions. The comet images were taken using a fluorescence microscope (Nikon Live Cell Imaging System). A total of 50 cells per treatment were analyzed, the comet tail length and tail percent DNA were calculated, and the mean value was determined. The images were processed by the automatic comet assay software CometScore Pro 2.0 (2012 TriTek Corp).
Live cell imaging system
Manufactured by Nikon
Sourced in United States, Japan
The Live Cell Imaging System is a laboratory equipment designed for the observation and analysis of living cells in real-time. It incorporates high-resolution microscopy, environmental control, and imaging capabilities to enable researchers to study cellular dynamics and processes in a controlled and non-invasive manner.
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7 protocols using live cell imaging system
1
Alkaline Comet Assay for Bortezomib
2
Bortezomib-Induced Mitotic Disturbance in T-ALL
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The mitotic disturbance induced by bortezomib treatment was studied in T-ALL cell lines after treatment with vehicle control and bortezomib for 24 h by using the previously published protocol with few modifications [61 (link)]. The cells were seeded in a density of 0.3 × 106 cells/mL (CCRF-CEM) or 1 × 106 cells/mL (PEER and LOUCY) and treated for 24 h along with the vehicle control. For PEER and LOUCY, the plates were coated with Poly-L-Lysine (0.1% w/v, Catalog No. P 8920, SIGMA-ALDRICH CO) prior to seeding the cells. The images of DAPI-stained nuclei were taken using a fluorescence microscope (Nikon Live Cell Imaging System) and were processed using ImageJ software 1.53t. A total of 300 cells from each group were analyzed, and the number of nuclei exhibiting the mitotic catastrophe was counted.
3
Topical and Oral SWT Inhibits DMBA-Induced Skin Carcinogenesis
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All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and approved by the Western University of Health Sciences Institutional Animal Care and Use Committees. Five-week-old female SENCAR mice (National Cancer Institute, Frederick, MD, USA) were divided into six groups (n = 6 or 8) and the backs of mice shaved. At seven weeks of age, 100 nmol DMBA dissolved in 200 µL acetone was applied topically twice weekly for four weeks. SWT treatment started when mice were five weeks of age, twice weekly, topically in two doses (0.64 and 1.28 mg/mL in 200 µL acetone) 30 min before DMBA exposure, or orally by gavage in two doses (200 and 1000 mg/kg in 1% methyl cellulose in PBS) 2 h before DMBA exposure. Two days after the last treatment mice were sacrificed, and samples of skin were excised and fixed immediately in formalin and embedded in paraffin blocks. The embedded tissues were cut into 3-micron thick sections and stained with H&E to determine the morphology. The images were obtained by EVOS; the epidermal thickness was measured using a Nikon Live-Cell Imaging system (Melville, NY, USA).
4
Oxidative Stress Response in Young and Old hMDSCs
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For oxidative stress experiments, 2 × 103 young or old donor hMDSCs were seeded in 24-well plates and cultured at 37 °C with 5% carbon dioxide overnight in proliferation medium. The following day, the proliferation medium was removed and cells were rinsed with PBS one time, and then, oxidative stress medium was added to four wells, each containing 500 or 650 μM H2O2 and propidium iodide (PI; 2 μg/ml). Only dead cells became intercalated with PI, which results in red fluorescence. The plates were set up in a NIKON live-cell imaging system. Four locations (fields of view) from each well were randomly chosen for image capture. Bright-field and red fluorescence images were taken every 10 min, and the number of dead cells was calculated at 0, 4, 8, 12, 16, 20, and 24 h using ImageJ software. The cell survival rates for young and old hMDSCs were calculated for each time point.
5
Fluorescent Labeling of Metaphase II Oocytes
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Metaphase II oocytes were incubated in DPBS containing 2.5% pronase for 2–3 min in order to remove their zona pellucida (ZP). Then, ZP-free oocytes were incubated with ER-Tracker Red (1:500) (Beyotime Institute of Biotechnology, China) or Mito-Tracker Green (1:1,000) (Beyotime Institute of Biotechnology, China) at 39°C, 5% CO2 for 30 min. Oocytes were washed three times and imaged using a Live Cell Imaging System (Nikon A1, Nikon, Tokyo, Japan). For live imaging, oocytes were isolated under mineral oil and imaged using A1, scanning the Z-axis of oocytes with the same depth (10 μm) and seven steps, and the maximum intensity projections of the equatorial cross-section of the oocytes were showed.
6
Tracing LIPTER RNA in Cardiomyocytes
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To trace LIPTER RNA in live CMs, LIPTER-24xMS2SL and LIPTER-antisense-24xMS2SL were separately inserted into a pHAGE lentiviral vector with puromycin resistance. The pHR-tdMCP-YFP (MS2-coat binding protein tagged with YFP) lentiviral plasmid was obtained d from Addgene (plasmid number 99151). A total of 1 × 106 hiPSCs were infected with packaged pHAGE-LIPTER-24xMS2SL or pHAGE-LIPTER-antisense-24xMS2SL lentivirus for 72 h. After infection, cells were selected using 1 µg ml−1 puromycin for 4 days. The remaining cells were then infected with pHR-tdMCP-YFP lentivirus. YFP+ cells were sorted by a BD FACSAria II Cell Sorter, expanded and differentiated into CMs. The differentiated CMs were seeded into poly-d -lysine-coated 35 mm glass-bottom dishes (MatTek, P35GC-0-10-C). Live cell video was taken on a Nikon live cell imaging system with an Apo 60× Oil lens at Indiana Center for Biological Microscopy. Consecutive images were taken during 4 h with 3 min intervals, and a total of 81 images were taken per field. The video was exported with 16 frames per second using the NIS Elements software.
7
Visualizing Lipid Droplet Accumulation in Bovine Preadipocytes
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Bovine preadipocytes were differentiated as described previously (Wang et al., 2018). Following differentiation for 4 or 8 days, the cells were visually analyzed using BODIPY or Oil red O (ORO) staining of the lipid droplets (LDs), respectively. For the ORO staining, the cells were washed 3 times with PBS and fixed with 4% paraformaldehyde for 30 min. They were stained with ORO solution (0.3% ORO, 60% isopropanol, and 40% PBS) for 30 min in the dark. For the BODIPY staining, the cells were incubated for 30 min after fixation, in a 1:1,000 dilution of 1 mg/ml BODIPY 493/503 (Invitrogen) in PBS at room temperature. We used 4,6-diamidino-2-phenylindole (DAPI) to identify the nuclei. Images were visualized with a Live Cell Imaging System (Nikon Instruments, Europe BV, Kingston, Surrey, England). Lipid accumulation was quantified from the fluorescence intensity parameter of the BODIPY dye (green fluorescence) using ImageJ software.
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