Ethanol

Manufactured by Beyotime

Sourced in China

Ethanol is a clear, colorless liquid that is commonly used in various laboratory applications. It serves as a solvent and disinfectant, capable of dissolving a wide range of organic compounds. Ethanol has a characteristic odor and exhibits a high flammability.

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13 protocols using ethanol

Cell Cycle and Apoptosis Analysis

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For cell cycle assay, A549/Taxol and H1299/Taxol cells were collected after transection and fixed in ethanol (70%; Beyotime) for 12 h at −20℃. After fixation, the cells were harvested via centrifugation, followed by addition of propidium iodide (PI; KeyGene) and RNase A (Beyotime). FACScan^® flow cytometry was employed to detect cell cycle distribution.
For detection of cell apoptosis, the transfected cells were harvested. After suspending in binding buffer (0.5 ml), cells were labeled with Annexin V‐FITC and PI (Beyotime), followed by measurement of apoptotic cells with FACScan^® flow cytometry.

Wu Y., Xie J., Wang H., Hou S, & Feng J. (2022). Circular RNA hsa_circ_0011298 enhances Taxol resistance of non‐small cell lung cancer by regulating miR‐486‐3p/CRABP2 axis. Journal of Clinical Laboratory Analysis, 36(5), e24408.

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Cell Cycle and Apoptosis Analysis

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For analysis of cell cycle progression, DU145/DTX and PC3/DTX cells were collected, followed by fixing with ice-cold ethanol (75%, Beyotime) at −20°C for 12 h. Next, the fixed cells were subsequently incubated with propidium iodide (PI; Sangon Biotech, Shanghai, China) and RNase A (Sangon Biotech) in the darkness for 0.5 h. The cells were analyzed by flow cytometry (BD Biosciences, Franklin, NJ, USA) for determination of cell cycle distribution. The apoptosis assay was conducted with the Annexin V-FITC/PI apoptosis detection kit (Sangon Biotech) via suspending the cells in binding buffer (400 μL) containing Annexin V-FITC and PI, incubating them in the darkness for 20 min. Finally, flow cytometry was utilized to quantify cell apoptosis.

Zhang H., Li M., Zhang J., Shen Y, & Gui Q. (2021). Exosomal Circ-XIAP Promotes Docetaxel Resistance in Prostate Cancer by Regulating miR-1182/TPD52 Axis. Drug Design, Development and Therapy, 15, 1835-1849.

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Transwell Migration and Invasion Assay

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Transwell assay was performed by transwell inserts (8 μm pore-size, (8 mm pores; Corning, Costar, NY, USA) either without Matrigel (for migration assay) or with Matrigel (for invasion assay) (BD Biosciences). Briefly, the serum-free media containing transfected cells was added to the top chamber, and the medium with 10% FBS was added to the lower part of the chamber. The cells on the bottom membrane surface were fixed in ethanol (Beyotime) and stained by crystal violet (Beyotime) after 24 h of incubation. Images were captured under a microscope (Leica, Wetzlar, Germany) at ×100 magnification.

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Cell Cycle and Apoptosis Analysis

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For analysis of cell cycle progression, cells were collected after treatment for 48 h, and then fixed with 70% ice-cold ethanol (Beyotime) at −20°C for 12 h. Next, the fixed cells were subsequently incubated with propidium iodide (PI; Sangon Biotech) and RNase A (Sangon Biotech) in the darkness for 15 min. At last, cell cycle distribution was determined by flow cytometer (Partec AG, Arlesheim, Switzerland). For the cell apoptosis analysis, cells were collected at 48 h post-treatment, and resuspended in 400 μL of binding buffer. Next, the cells were then incubated with 5 µL of Annexin V-fluorescein isothiocyanate (FITC) and 10 µL of PI (Sangon Biotech) in the darkness for 20 min. The apoptotic cells were analyzed by flow cytometry.

Li L., Jiang Z., Zou X, & Hao T. (2021). Exosomal circ_IFT80 Enhances Tumorigenesis and Suppresses Radiosensitivity in Colorectal Cancer by Regulating miR-296-5p/MSI1 Axis. Cancer Management and Research, 13, 1929-1941.

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Cell Proliferation Assays for PC3 and DU145 Cells

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Cell proliferation was determined by cell counting Kit-8 (CCK-8), colony formation, and 5-Ethynyl-2′-Deoxyuridine (EDU) incorporation assays. For CCK-8 assay, transfected PC3 and DU145 cells were placed into 96-well plates with 3 × 10³ per well. At 24, 48, or 72 h incubation, each well was added with 10 μL of CCK-8 reagent (Beyotime, Shanghai, China) and incubated for another 1 h. The absorbance in each well was measured using a microplate reader at 450 nm (Bio-Rad, Hercules, CA, USA). For colony formation assay, PC3 and DU145 following assigned transfection were plated at 6-well plates (500 cells/well). After culture for 14 days, cell colonies were fixed with ethanol (Beyotime), and the number of colonies was imaged and counted after crystal violet staining. For the EDU incorporation assay, an EDU incorporation assay kit (RiboBio, Guangzhou, China) was used. Transfected PC3 and DU145 cells were incubated with 50 mM EDU for 2 h. Then, cells were fixed with 4% paraformaldehyde (Beyotime) and stained with Apollo Dye Solution for proliferating cells. Subsequently, the nuclei were stained with DAPI. The images were obtained employing a fluorescence microscope, and stained cells were analyzed using ImageJ software (National Institutes of Health, Sacaton, AZ, USA).

Qin L., Sun X., Zhou F, & Liu C. (2021). CircLRP6 contributes to prostate cancer growth and metastasis by binding to miR-330-5p to up-regulate NRBP1. World Journal of Surgical Oncology, 19, 184.

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Colony Formation Assay of Transfected Cells

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Transfected cells (DU145/DTX and PC3/DTX) were plated into 12-well plates. The cells were then cultured for 14 days, and the medium was replaced as needed. After that, the colonies were fixed with ethanol (75%, Beyotime), followed by staining with violet (0.1%, Beyotime). At last, colonies (>50 cells/colony) were counted and photographed.

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Colony Formation and Transwell Invasion Assay

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The transfected cells were seeded in a 6‐well plate for 14 days. The colonies were fixed with ethanol (75%, Beyotime), followed by staining with violet (0.1%, Beyotime). Finally, colonies (>50 cells/colony) were counted and photographed.
The transfected cells were resuspended with serum‐free medium and seeded in the transwell upper chamber. Then, 500 μl of DMEM medium supplemented with 10% FBS was added to the transwell lower chamber. After incubation for 48 h, the chambers were stained with 0.5% crystal violet and counted under a Nikon microscope (Minato).

Li J., Zhu T., Weng Y., Cheng F., Sun Q., Yang K., Su Z, & Ma H. (2022). Exosomal circDNER enhances paclitaxel resistance and tumorigenicity of lung cancer via targeting miR‐139‐5p/ITGB8. Thoracic Cancer, 13(9), 1381-1390.

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Cell Cycle Analysis of Leukemic Stem Cells

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CD34+CD38− LSCs were harvested and washed twice in PBS. CD34+CD38− LSCs were fixed overnight using 70% ice-cold ethanol (Beyotime Biotech., Shanghai, China), and washed twice using PBS. Then, the CD34+CD38− LSCs were treated using bovine pancreatic ribonuclease (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 1 mg/ml for 30 min at 37°C. The cells were stained with the propidium iodide (PI) at a concentration of 50 μg/ml in the dark for 30 min. The stained CD34+CD38− LSCs were viewed and imaged using the FACS Aria IIU flow cytometer (BD Bioscience, Oxford, UK) and the percentages of cells in each phase of the cell cycle were analyzed using MultiCycle software (Phoenix Flow Systems, Tokyo, Japan).

Tang Y.L., Zhang C.G., Liu H., Zhou Y., Wang Y.P., Li Y., Han Y.J, & Wang C.L. (2020). Ginsenoside Rg1 Inhibits Cell Proliferation and Induces Markers of Cell Senescence in CD34+CD38− Leukemia Stem Cells Derived from KG1α Acute Myeloid Leukemia Cells by Activating the Sirtuin 1 (SIRT1)/Tuberous Sclerosis Complex 2 (TSC2) Signaling Pathway. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e918207-1-e918207-8.

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Quantifying Neuronal Apoptosis via Nissl Staining

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Nissl staining assay was performed to observe neuronal cell death in brain sections. First, brain paraffin sections were de-paraffinized with xylene and then dehydrated in a graded concentration of ethanol (70, 80, 90 and 100%; Beyotime Institute of Biotechnology). Then the brain paraffin sections were placed in 0.2% Nissl staining solution for 5 min at room temperature. Representative images of Nissl-stained brain sections in the cortex and CA1 and CA3 regions on day 7 following cerebral ischemia were captured under a high-power light microscope (magnification, ×200). The number of apoptotic neurons was counted using ImageJ software.

Wang Y., Cai X., Wu Z., Tang L., Lu L., Xu Y, & Bao X. (2021). Tetrandrine attenuates ischemia/reperfusion-induced neuronal damage in the subacute phase. Molecular Medicine Reports, 23(4), 297.

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Hippocampal Nissl Staining Protocol

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Hippocampal tissue 2.64 to 5.40 mm posterior to the bregma was selected, de-waxed, hydrated, and stained with Nissl staining solution (Shanghai Beyotime Biotechnology Co., Ltd, Shanghai, China) for 10 min. After ethanol (Beyotime) dehydration and washing with distilled water, sections were cleared with xylene (Beyotime), sealed with neutral gum (Beyotime), and observed under the microscope (Olympus Optical Co., Ltd, Tokyo, Japan). The number of Nissl-stained cells was then counted.

Wu Z.S., Huang W.L, & Gong S.J. (2019). Effect of adenovirus-mediated overexpression of PTEN on brain oxidative damage and neuroinflammation in a rat kindling model of epilepsy. Chinese Medical Journal, 132(21), 2628-2635.

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