Followin the indicated treatment, newly synthesized DNA in U-251MG and U-87MG cells was measured by EdU fluorescence staining based on the manufacturer’s protocol (Click-iT® EdU Imaging Kits, Invitrogen). The cells, cultured in 96-well plates at a density of 8 × 103 cells/well, were labeled with 10 μM EdU, incubated for 3 h, and then fixed for 20 min with 3.7% formaldehyde at room temperature. Then, the fixative was removed, and the cells in each well were washed three times with 3% BSA in PBS. The BSA solution was removed, and the cells were permeabilized with 0.5% Triton X-100 (Sigma, San Francisco, CA, USA) for 20 min at room temperature. After washing the cells three times with 3% BSA in PBS, a 100 μL of Click-iT® reaction cocktail was added to each well, and the plate was incubated for 30 min at room temperature in the dark. Then, 1 mL of Hoechst 33342 nuclear staining solution (Sigma, San Francisco, CA, USA) was added to each well, and the plate was incubated for 25 min at room temperature in the dark. Subsequently, the staining solution was removed, and the cells were washed three times with PBS. Then, the EdU-labeled cells were photographed and counted using a fluorescence microscope (CKX41-F32FL, Olympus, Tokyo, Japan). Image-Pro Plus software (Version 5.0, MD, USA) was used to determine the percentage of EdU-positive (EdU+) cells.
Ckx41 f32fl
Manufactured by Olympus
Sourced in Japan, China
The CKX41-F32FL is a compact fluorescence inverted microscope designed for routine laboratory applications. It features a fluorescence illumination system and a wide range of optical accessories to support various microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Click it edu imaging kit, by Thermo Fisher Scientific (9 mentions)
Triton x 100, by Merck Group (7 mentions)
Hoechst 33342 nuclear staining solution, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole solution, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Click it reaction cocktail, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Alexa fluor plus 594, by Thermo Fisher Scientific (1 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Caspase 1, by Proteintech (1 mentions)
Nlrp3, by Proteintech (1 mentions)
Goat serum, by Beyotime (1 mentions)
Nsc23766, by Bio-Techne (1 mentions)
20 protocols using ckx41 f32fl
1
Quantifying DNA Synthesis in Glioma Cells
2
Characterization of Self-Healing Bitumen Microcapsules
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The dried microcapsules were observed by using an Environmental Scan Electron Microscopy (ESEM, Philips XL30, Philips, Amsterdam, The Netherlands) at an accelerated voltage of 20 kV. Self-healing behaviors of bitumen were observed by a fluorescence microscope (CKX41-F32FL, OLYMPUS, Tokyo, Japan). As bitumen is a temperature-sensitive material, the observation is in an environment of 0 °C temperature.
About 2 g MMF-shell microcapsules was mixed in 5 g epoxy resin. After the composite was dried at room temperature, it was carefully cut to obtain the cross-section. The thickness of shells can be measured from the SEM images of cross-section of microcapsules [4 (link)]. At least 20 shells of each microcapsule sample were measured and the average data were calculated.
About 2 g MMF-shell microcapsules was mixed in 5 g epoxy resin. After the composite was dried at room temperature, it was carefully cut to obtain the cross-section. The thickness of shells can be measured from the SEM images of cross-section of microcapsules [4 (link)]. At least 20 shells of each microcapsule sample were measured and the average data were calculated.
3
EdU Assay for Proliferating Cells
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The EdU assay was conducted according to previous study [40 (link)]. The cells were pre-inoculated in a 24-well plate at a density of 5 × 103 cells per well. The plated cells were then incubated in 4% methanol for 30 min, followed by permeabilization in 0.5% TritonX-100 for 10 min, and a 30-min reaction in 400 μL of 1× ApollorR. Afterward, the cells were stained with 4ʹ,6-diamidino-2-phenylindole for another 30 min. The EdU-stained cells were counted under a fluorescence microscope (CKX41-F32FL, Olympus, Tokyo, Japan) to calculate the percentage of EdU-positive cells.
4
Apoptosis Quantification in H9c2 Cells
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The apoptosis of H9c2 cells was measured by Hoechst 33342/PI staining Kit (GENVIEW) following the manufacturer’s instructions. H9c2 cells were harvested and washed three times with PBS after IH stimulation. Then the cells were stained by incubating in staining buffered solution containing 500 nM Hoechst 33342 and 500 nM PI at RT for 15 min in the dark. Finally, they were washed with PBS three times and observed under a fluorescence microscope (Olympus, CKX41-F32FL, Japan).
5
GFP Expression in RPMI-8226 Cells
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Recombinant plasmids containing the green fluorescent protein (GFP) gene which expresses GFP, and the transfection efficiency may be directly observed under an inverted fluorescence microscope (CKX41-F32FL, Olympus, Tokyo, Japan). RPMI-8226 cells were seeded in six-well plates overnight and then transfected with siRNA or negative control siRNA oligonucleotides (containing the GFP gene which emits green light) that were precomplexed with Lipofectamine 2000 (Invitrogen Life Technologies). The medium was refreshed after 6 h with complete growth medium, and the cells were incubated for an additional 48 h. Following this, antibiotic selection (0.2 μg/ml puromycin; Invitrogen Life Technolgies) was initiated and continued for 14–20 days prior to selection of stably transfected cells. The siRNA sequences used to target DNMT1 were 5′-CACTGGTTCTGCGCTGGGA-3′ (sense) and 5′-AAGTCTTCTGACGCTGCTGCCTGGTCCAG-3′ (antisense), and were designed based on GenBank accession no. NM_001379.1.
6
Evaluating Apoptosis in Cancer Cells
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The cells were cultured in six-well plates in RPMI 1640 supplemented with 10% FBS medium, and were treated with DOX and CXB alone or with a combination of CXB/DOX for 24, 48 and 72 h. The cover slips were washed three times with phosphate-buffered saline (PBS) and single cell suspensions were fixed in 1% PBS. The cells were stained with 100 μg/ml acridine orange and 100 μg/ml ethidium bromide for 1 min. The cells were then observed under a fluorescence microscope (CKX41-F32FL, Olympus, Tokyo, Japan). At least 200 cells were counted and the percentage of apoptotic cells was determined. Triplicates were performed in all experiments and each experiment was performed three times.
7
Quantifying H9c2 Cell Proliferation
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The proliferation of H9c2 cells was detected by BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 following the manufacturer’s instructions. In brief, H9c2 cells were seeded in 12-well plates and incubated with 100 µl of 50 µM EdU for 2 h after IH stimulation. And then the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Trixon-100 for 5 min. According to the kit, the cells were incubated in the mixture of Click Reaction Buffer, CuSO4, Azide 594, and Click Additive Solution for 30 min at RT. After three times washes in PBS, the cell nucleus was stained with 100 µl DAPI for 20 min. The staining cells were observed using a fluorescence microscope (Olympus, CKX41-F32FL, Japan).
8
Measuring DNA Synthesis by EdU Assay
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DNA synthesis was performed by a 5-ethynyl-2´-deoxyuridine (Edu) incorporation assay (Click-iT® EdU Imaging Kits, invitrogen, USA) according to the manufacturer’s instructions. Briefly, cells were incubated with Edu-labeling solution for 2h at 37°, and then the cells were fixed with 4% cold formaldehyde for 30 min at room temperature. After permeabilization with 1% Triton X-100, the cells were reacted with Click-iT® reaction cocktails (invitrogen) for 30 min. Subsequently, the DNA contents of the cells were stained with Hoechst 33342 for 30 min. Finally, Edu-labeled cells were counted using fluorescence microscopy (CKX41-F32FL, Olympus) and normalized to the total number of Hoechst-stained cells.
9
Evaluating miR-3148 in Glioma Cell Proliferation
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After culturing cells at 37˚C for 24 h in a 96-well plate at a density of 2x103 cells/well, U87 MG and U251 cell lines were incubated at 37˚C for 2 h with 10 µl Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) solution. Then, the optical density at 450 nm was measured and recorded.
The role of miR-3148 in glioma cell proliferation was evaluated using Click-iT® EdU Imaging Kits (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. U87 MG and U251 cells were cultured in 96-well plates (8x103 cells/well) and incubated at room temperature with 10 µl of EdU reagent for 3 h. At room temperature, the cells were fixed with 4% formaldehyde for 20 min and washed with formaldehyde in PBS. The cells were then incubated at room temperature in 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 5 min. The nuclei were then stained with a 4',6-diamidino-2-phenylindole solution (1 ml; Sigma-Aldrich; Merck KGaA), which was added to each well and incubated for 25 min at room temperature in the dark. The staining solution was then removed by washing thrice with PBS. Finally, the stained cells were imaged and counted under a fluorescence microscope (magnification, x100) (CKX41-F32FL; Olympus, Beijing, China). Each assay was performed in triplicates.
The role of miR-3148 in glioma cell proliferation was evaluated using Click-iT® EdU Imaging Kits (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. U87 MG and U251 cells were cultured in 96-well plates (8x103 cells/well) and incubated at room temperature with 10 µl of EdU reagent for 3 h. At room temperature, the cells were fixed with 4% formaldehyde for 20 min and washed with formaldehyde in PBS. The cells were then incubated at room temperature in 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 5 min. The nuclei were then stained with a 4',6-diamidino-2-phenylindole solution (1 ml; Sigma-Aldrich; Merck KGaA), which was added to each well and incubated for 25 min at room temperature in the dark. The staining solution was then removed by washing thrice with PBS. Finally, the stained cells were imaged and counted under a fluorescence microscope (magnification, x100) (CKX41-F32FL; Olympus, Beijing, China). Each assay was performed in triplicates.
10
Mesangial Cell Morphology Analysis
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The mesangial cells morphology was observed using an inverted microscope (CKX41-F32FL, Olympus). The observed parameter included cells proliferation that was indicated by the density of the cells. The morphology of the treated mesangial cell group was compared to the non-treated mesangial cell as the control group.
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