Cells were seeded in 6- or 12-well plates, containing coverslips, in 1–3 mL of complete medium. Medium was changed and cells were treated 24 hours after seeding. For the study of cells in S-phase, EdU 10 μmol/L (5-ethynyl-2′-deoxyuridine, Thermo Fisher Scientific) was added for 10 (MIA PaCa-2), 15 (Capan-1) or 20 minutes (BxPC3). Cells were pre-extracted with 0.2% Triton-X100 for 3 minutes, fixed in 4% PFA for 15 minutes and permeabilized with 0.5% Triton X-100 for 20 minutes. For revealing EdU, the Click-iT EdU Imaging kit (Thermo Fisher Scientific) was used. Samples were blocked with PBS 5% BSA. Primary antibodies were anti–phospho-histone H2AX (Ser139; JBW301, Millipore 05–636, 1:1,000), anti–phospho-53BP1 (S1778; Cell Signaling Technology #2675, 1:1,000), anti–phospho-RPA2 (S4-S8; Bethyl A300–245A, 1:500), anti-RPA70 (Cell Signaling Technology #2267, 1:500), anti-FANCD2 (NOVUSBIO NB100–182, 1:1,000), and anti-53BP1 (Bethyl A300–272A, 1:2,500). DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) and images acquired on Nikon DS-Qi2 fluorescence microscope. ImageJ and CellProfiler software were used to quantify the number of foci and staining intensity per nucleus. For each condition, at least 500 cells were measured. For quantification in early mitotic cells, at least 50 cells in prophase and prometaphase were counted.
Ds qi2 fluorescence microscope
Manufactured by Nikon
Sourced in Japan
The DS-Qi2 is a fluorescence microscope designed for high-quality imaging. It features a 16.25-megapixel CMOS sensor and supports a variety of fluorescence imaging techniques, including DAPI, FITC, TRITC, and Cy5. The microscope is capable of capturing images with a maximum resolution of 4920 x 3264 pixels.
Automatically generated - may contain errors
2 protocols using ds qi2 fluorescence microscope
1
Quantifying DNA Damage Response Foci
2
Zebrafish Larval Toxicity Assay
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The zebrafish larvae from 96hpf after fertilization were randomly put into a 12-well plate with 20 zebrafish in each well. 4 mL of the PAR liquid was added to each well, and 3 replicates were set in the experiment. The 12-well plate was placed in the standard culture environment and kept at 28.5 ± 0.5°C for 14/10 h light-dark cycle. After PAR treatment for 24 hours, the death of zebrafish in each experimental group was observed and recorded under a DS-Qi2 fluorescence microscope (Nikon, Japan), and the average mortality of each group was calculated. SPSS20.0 was used to draw the best “mortality-concentration” effect curve, calculate the lethal concentration of the 10% zebrafish larvae (LC10), and used the LC10 concentration in the rest of the experiments to provide a reference for the concentration setting of the target organ identification test.
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