The cell culture and p53 transfection was conducted as described in section “Real-time quantitative PCR (RT-qPCR).” The cells were digested with 0.25% trypsin solution and reseeded into 6-well plates at a density of 1.0×104 cells/well. After one week of the culture, the cells were treated with 70% cold ethanol at 4°C for 20 minutes and stained with 0.2% crystal violet solution, followed by the observation to record the colony formation under an IX73P1F fluorescence microscopy (Olympus Corporation). Finally, 33% acetic acid solution was employed to treat the cells, and the optical density at 578 nm was measured using GF-M3000 microplate reader.
Ix73p1f fluorescence microscopy
Manufactured by Olympus
Sourced in Japan
The IX73P1F is a fluorescence microscope designed for biological and material science research. It features a motorized focusing mechanism, LED illumination, and a high-resolution camera interface for capturing detailed images of fluorescently labeled samples.
Automatically generated - may contain errors
10 protocols using ix73p1f fluorescence microscopy
1
Cell Culture, p53 Transfection, and Colony Formation Assay
2
Evaluating Doxorubicin-Induced Skin Damage
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All the animal studies were conducted according to the “Guide for the Care and Use of Laboratory Animals” (8th edition, International Publication No: 978-0-309-15,400-0) and approved by the Institutional Animal Ethics Committee of Jilin University (license No. SCXK-(JI) 2014–0012). Six-week-old Wistar female rats were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Before use in this study, all rats were provided with standard diet and water for one week. To construct the HFS animal models, DOX or PLD (10 mg/kg) was intravenously injected into the Wistar rats once every 3 days. The onset of HFS was recognized as the redness of the skin. The rats were weighed every day and sacrificed on day 10. The paws of rats were photographed after 1 minute and 10 days since the administration. Subsequently, the skin of the damaged paws and main organs were collected, fixed in 10% formalin solution, dehydrated and then embedded in paraffin. The embedded tissues were sectioned into 4-μm slides, which were stained with hematoxylin and eosin (H&E) solution and observed by Olympus IX73P1F fluorescence microscopy. Meanwhile, to detect the dermal collagen fibers in the skin, the sections were stained with picrosirius red and then photographed with a NIKON Eclipse ci polarized light microscopy (Tokyo, Japan).
3
Apoptosis and Viability Evaluation of Nanoparticles
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BMDMs and RAW264.7 cells were seeded in 6-well plates (3.0 × 105 cells/well) and stimulated with 0.1 μg/ml LPS for 24 h. The transfection was conducted using different nanoparticles in FBS-free medium for 6 h, and the cells were further incubated in 10% FBS-containing medium for 48 h. For the apoptotic ratio analysis, the cells were treated with Annexin V-FITC and PI solutions according to the manufacturer’s instructions and detected by CytoFLEX flow cytometry system (Beckman Coulter, Brea, CA). The data were analyzed by CytExpert 2.3. For the live/dead cell staining, the cells were washed with PBS three times and stained with 200 μl combined live/dead cell staining solution provided in the detection kit. For the TUNEL staining, the cells were fixed in 4% (w/v) paraformaldehyde solution for 30 min and then treated with 0.3% (v/v) Triton X-100 for 5 min, and then stained with TUNEL reagent at 37 °C for 1 h. After rinsing with PBS three times, the cells were subjected to the analysis on Olympus IX73P1F fluorescence microscopy (Tokyo, Japan) to acquire the images.
4
HepG2 Cell Viability Assay with Nanoparticles
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The HepG2 cells were inoculated in 6-well plates at a density of 2.5×105 cells/well and cultured at 37°C overnight. Then, the cells were treated with PP/Dz13 (mass ratio of 10.0) and PAMAM/Dz13 nanoparticles (mass ratio of 7.5) containing 5 μg/mL Dz13 in serum-free medium for 6 hrs and further in 10% FBS-containing DMEM for 48 hrs. Based on the manufacturer’s instructions, live/dead reagents were used to stain the cells which were observed by Olympus IX73P1F fluorescence microscopy (Tokyo, Japan).
5
Tissue Apoptosis Detection Protocol
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The sectioned skin tissues were first incubated with 0.25% of proteinase K solution at room temperature for 15 min, and washed with PBS twice. Then 0.1% Triton X-100 solution was added to completely cover the sections which were incubated at room temperature for 2 min. Afterwards, the sections were treated with TUNEL solution and covered with coverslips according to the manufacturer’s instructions. Finally, the sections were placed in the dark for 60 min and observed on an Olympus IX73P1F fluorescence microscopy (Tokyo, Japan).
6
TUNEL Assay for Apoptosis Detection
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The cell culture and the treatment with DOX or PLD were carried out as described in the section “Cell apoptosis analysis”, then the cells were rinsed with PBS three times and treated with 0.1% Triton X-100 solution at room temperature for 2 min. After being rinsed with PBS, the cells were treated with TUNEL reagent according to the manufacturer’s protocol. Finally, the cells were rinsed with PBS again and observed on an Olympus IX73P1F fluorescence microscopy (Tokyo, Japan).
7
Mitochondrial Membrane Potential Analysis
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The cell culture and p53 gene transfection was carried out as described in section “Real-time quantitative PCR (RT-qPCR),” and the mitochondrial membrane potential changes were detected using JC-1 probe. Typically, the collected cells were dyed with JC-1 probe provided in the detection kit at 37°C for 30 minutes. The mitochondrial membrane potential was finally examined by IX73P1F fluorescence microscopy (Olympus Corporation) after the cells were rinsed with PBS twice.
8
Nanoparticle-Mediated Apoptosis and Cell Cycle Arrest
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In the cell apoptosis analysis, HepG2 cells were inoculated into 6-well plates at an initial density of 1.5×105 cells/well, and transfected with different nanoparticles (N/P ratio of 30, 0.5 μg/mL siPlk-1) in FBS-free DMEM for 6 h. Afterwards the cells were cultured in 10% FBS-containing DMEM at 37 °C for 48 h, and stained with Annexin V-FITC and PI according to the apoptosis detection kit’s instructions. For the cell cycle arrest analysis, the transfection of siPlk-1 was conducted as described above, and the cells were resuspended in PBS, fixed by 75% ethanol, and stained with PI at 4 °C for 30 min. Finally, the cell apoptosis and cell cycle arrest were detected using CytoFLEX flow cytometer (Beckman Coulter Inc., CA). To analyze the mechanism of cell apoptosis, the cells after the transfection of siPlk-1 were washed with PBS three times and stained with JC-1 probe at 37 °C for 20 min according to the mitochondrial membrane potential assay kit’s instructions. The images were captured by IX73P1F fluorescence microscopy (Olympus, Tokyo, Japan).
9
Evaluating Anti-Migration Effects of siPlk-1
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The anti-migration effect after siPlk-1 transfection was evaluated by wound healing and Transwell migration assays. In the wound healing assay, HepG2 cells were seeded into 6-well plates at a density of 2.5×105 cells/well and cultured for 24 h. The wound was then generated in each well by scratching using a 200-µL pipette tip, after which the cells were treated with different nanoparticles in DMEM for 6 h. The width of wound was measured and photographed using IX73P1F fluorescence microscopy (Olympus, Tokyo, Japan) at different time points. In the Transwell migration assay, HepG2 cells after the transfection with different nanoparticles were inoculated into the upper chamber of 24-well Transwell (2.0×104 cells), and the lower chamber was filled with 700 µL DMEM containing 10% FBS. After the culture at 37 °C for 24 h, the chambers were washed with PBS three times, and the cells on the upper surface of membrane were removed carefully. The cells on the lower surface were fixed using 75% ethanol at 4 °C for 20 min and then stained by 0.2% crystal violet at 25 °C for 15 min. Finally, the cells were observed using IX73P1F fluorescence microscope (Olympus, Tokyo, Japan), and the absorbance at 570 nm was measured by HBS-1096A microplate reader (Detie, Nanjing, China) to quantify the cells transferring across the membrane.
10
Live/Dead Cell Viability Assay
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The cell culture and p53 transfection was conducted as described in section “Real-time quantitative PCR (RT-qPCR).” The collected cells were rinsed with 500 µL PBS twice and subjected to the staining at room temperature for 30 minutes with 200 µL of live/dead reagent composed of 2 mM calcein AM and 4 mM ethidium homodimer. The images were obtained through the observation under IX73P1F fluorescence microscopy (Olympus Corporation, Tokyo, Japan).
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