A certain dose of ANGPTL4 was used to treat 1 × 105 BMSCs, cultured in 12 well plate with serum‐free DMEM for 24 h, centrifuged at 1500 g for 5 min, and washed twice with 1 × PBS. The cells were fixed with 70% ethanol (Solarbio, Beijing), placed at 20°C for 15 min, centrifuged at 1500 g for 5 min, and washed twice by precooling 1 × PBS. Subsequently, the cells were incubated with 10 μg/μl Dnase‐free RnaseA (Sigma, St. Louis, MO, USA) to remove RNA, and washed twice with precooled 1 × PBS. Finally, after centrifugation at 150 g for 5 min, the cells were incubated with 1 mg/ml iodide (Sigma) in the dark at 4°C for 12 min. Flow cytometry and ModiFit software (Olympus, Tokyo, Japan) were used to quantify the reactive oxygen species (ROS) level and glucose uptake of BMSCs. In addition, according to the instructions, annexin FITC/PI apoptosis detection kit (Beyotime, Nanjing, China) was combined with flow cytometry to detect apoptosis (Dong & Cui, 2017 ). ModiFit software (Olympus, Tokyo, Japan) was performed to analyze the data.
Ethanol
Manufactured by Solarbio
Sourced in China
Ethanol is a clear, colorless liquid chemical compound commonly used in various laboratory applications. It serves as a solvent, disinfectant, and fuel source in controlled environments.
Automatically generated - may contain errors
Lab products found in correlation
Iodide, by Merck Group (4 mentions)
Modifit software, by Olympus (3 mentions)
Annexin fitc pi apoptosis detection kit, by Beyotime (3 mentions)
Dnase free rnase a, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (2 mentions)
Ab 8 macroporous resin, by Solarbio (2 mentions)
Phosphate buffered saline (pbs), by Solarbio (2 mentions)
Sodium chloride (nacl), by Solarbio (2 mentions)
15 ml centrifuge tube, by Corning (1 mentions)
Annexin pi kit, by Keygen Biotech (1 mentions)
Acetone, by Solarbio (1 mentions)
Hydrochloric acid (hcl), by Solarbio (1 mentions)
Mgcl2, by Solarbio (1 mentions)
Formaldehyde, by Solarbio (1 mentions)
Potassium chloride (kcl), by Solarbio (1 mentions)
β mercaptoethanol, by Solarbio (1 mentions)
Glutaraldehyde, by Solarbio (1 mentions)
Flow cytometry, by Bio-Rad (1 mentions)
Fluorescein isothiocyanate (fitc), by Solarbio (1 mentions)
Annexin 5, by Solarbio (1 mentions)
20 protocols using ethanol
1
ANGPTL4 Modulates BMSC ROS and Apoptosis
2
Cell Cycle and Apoptosis Analysis
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A total of 1×105 SW480 or LoVo cells were cultured in 12-well plate by using serum-free DMEM for 24 h, centrifuged at 150 × g for 5 min, and washed twice with pre-cooled 1X PBS. Subsequently, the cells were fixed with 70% ethanol (Solarbio), maintained at 20°C for 15 min, centrifuged at 150 × g for 5 min, and washed twice with pre-chilled 1X PBS. Next, the cells were incubated with 10 µg/µl DNase-free RNaseA (Sigma-Aldrich; Merck KGaA) for 45 min at 37°C to eliminate RNA, and washed twice with pre-chilled 1X PBS. Finally, following centrifuging at 150 × g for 5 min, the cells were incubated with 1 mg/ml iodide (Sigma-Aldrich; Merck KGaA) in the dark at 4°C for 12 min. The cell distribution at each phase of the cell cycle was quantified in a flow cytometer and using ModFit software 3.3 (Verity Software House). In addition, according to the manufacturer's instructions, Annexin-FITC/PI Apoptosis Detection Kit (Beyotime Institute of Biotechnology) was combined with flow cytometry to detect cell apoptosis (19 (link)). ModFit software 3.3 was performed to analyze the data.
3
Cell Cycle Analysis and Apoptosis Assay
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A total of 1 × 105 chondrocytes were cultured in a 12-well plate using serum-free DMEM for 24 h, centrifuged at 1500 g for 5 min and washed with pre-cooled 1 × PBS twice. Subsequently, cells were fixed with 70% ethanol (Solarbio, Beijing, China), placed at 20 °C for 15 min, then centrifuged at 1500 g for 5 min, and washed twice with pre-chilled 1 × PBS. Subsequently, cells were incubated with 10 μg/μL DNase-free RNaseA (Sigma, St. Louis, USA) for 45 min at 37 °C to eliminate RNA, and washed twice with pre-chilled 1 × PBS. Finally, following centrifuging at 150g for 5 min, the cells were incubated with 1 mg/mL iodide (Sigma, St. Louis, USA) in the dark at 4 °C for 12 min. The percentage of cells at each cell cycle phase is quantified in a flow cytometer and analyzed by ModiFit software (Olympus, Tokyo, Japan). Besides, according to instructions, Annexin-FITC/PI Apoptosis Detection Kit (Beyotime, Nanjing, China) was combined with flow cytometry to detect cell apoptosis, according to manufacturer’s instructions [17 (link)]. In addition, modified software (Olympus, Tokyo, Japan) was performed to analyze the data.
4
SV40 MES13 Cell Cycle Analysis
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Cell cycles of SV40 MES13 cells were detected by flow cytometry. In detail, 1 × 10 5 SV40 MES13 cells were cultured in 12-well plates with serum-free DMEM for 24 h, centrifuged at 1,500 g for 5 min, and washed twice with pre-cooled 1 × PBS. The cells were then fixed with 70% ethanol (Solarbio), placed for 15 min, centrifuged at 1,500 g for 5 min, and washed twice with pre-cooled 1 × PBS. Cell distribution at each stage of the cell cycle was quantified in flow cytometers and Mod-iFit software (Olympus, Tokyo, Japan) after incubating for 12 min in the dark at 4°C with 1 mg/mL iodide (Sigma, St. Louis, MO, USA).
5
Fabrication of Ionically Crosslinked Microgels
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Fabrication of IGMs was based on the microfluidic chip technology. The aqueous phase comprised a solution containing GelMA (100, 150, or 200 mg/mL), chitosan (10 mg/mL), 0.5% w/v Irgacure 2959, and 2% v/v acetic acid. The oil phase was composed of mineral oil (anhydrous, ≥ 99.9%, Aladdin Biochemical Materials Technology Co., Ltd) and 5% v/v Span 80 (Aladdin Biochemical Materials Technology Co., Ltd). Two syringes, one containing the aqueous phase solution and the other, the oil phase solution, were placed horizontally on the injection pump (Xunfei Scientific Instruments Co., Ltd, Suzhou, China). The aqueous phase and oil phase solutions were pushed into two separate inlets at distinct flow rates so that water-in-oil droplets were formed and photo crosslinked in a UV irradiation area using a 365 nm handheld UV lamp (Zigu Lighting Appliance Factory) for 20 s before being allowed to flow into a 15-mL centrifuge tube (Corning, New York, USA) containing 1 mL of 0.5 M NaOH, to solidify the chitosan. The obtained IGMs were triple-washed with deionized water and centrifuged at 200 × g for 6 min to remove the excess solution as well as the mineral oil and Span 80. Ethanol (Solarbio, Beijing, China) was used to gradually dehydrate the IGMs. Finally, the sample was dried with a scC02 desiccator (Leica, Wetzlar, Germany) for 2 h and stored in a dryer.
6
Annexin-PI Apoptosis Assay in T24 and HT-1197 Cells
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Cell apoptosis was evaluated by an annexin/PI Kit (KeyGen). T24 and HT-1197 cells were washed by using PBS (Solarbio) and rehanged, then fixed in 70% cooled ethanol (Solarbio). Ultimately, the rate of apoptosis was analyzed by Flow Cytometer (BD Bioscience, Massachusetts, USA).
7
Tilapia Protein Extraction Protocol
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Fresh tilapia (500 g ± 100 g) was purchased from the Yonghui supermarket (a local supermarket in Fuzhou, China). Curdlan was purchased from VWR Life Science Amresco Products, Avantor Performance Materials, Inc. Tris, HCl, KCl, NaN3, β‐mercaptoethanol, Mg (CH3COO)2, ethylene glycol‐bis‐(2‐chloroethyl) tetraacetic acid (EGTA), ATPNa2, KHCO3, MgCl2, formaldehyde, glutaraldehyde, phosphoric acid salt buffer, ethanol, and acetone were obtained from Solarbio Science & Technology Co., Ltd. The above reagents were all of analytical grade.
8
Cell Cycle and Apoptosis Analysis
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Targeted cells were digested with 0.25% trypsin, and 1 × 10 6 cells were immobilized with 75% cold ethanol (Solarbio, Shanghai, China) for 12 h. Cells were incubated with 50 μg/mL RNA enzymes (Solarbio, Shanghai, China) at room temperature for 1 h followed by propidium iodide (PI) (Solarbio, Shanghai, China) staining for 30 min in the dark. Flow cytometry (Bio-Rad, California, USA) was used to analyze the cell cycle. Every test was performed in triplicate.
The harvested cells were counted, and 1 × 10 6 cells were mixed with Annexin V (Solarbio, Shanghai, China) labeled with fluorescein isothiocyanate (FITC) (Solarbio, Shanghai, China) and incubated at 37°C for 10 min. Following three PBS washes for 5 min, PI staining was conducted in the dark at room temperature for 15 min, and cell apoptosis was detected by Flow cytometry. The apoptosis rate is presented as the percentage of early apoptotic cells among total cells. Every test was performed in triplicate.
The harvested cells were counted, and 1 × 10 6 cells were mixed with Annexin V (Solarbio, Shanghai, China) labeled with fluorescein isothiocyanate (FITC) (Solarbio, Shanghai, China) and incubated at 37°C for 10 min. Following three PBS washes for 5 min, PI staining was conducted in the dark at room temperature for 15 min, and cell apoptosis was detected by Flow cytometry. The apoptosis rate is presented as the percentage of early apoptotic cells among total cells. Every test was performed in triplicate.
9
Topotecan and Carvacrol Cytotoxicity Assay
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Topotecan (TOPO) hydrochloride and carvacrol were purchased from Sigma Aldrich Co. TOPO stock solutions were prepared by dissolving it in distilled water (D.W) and preserved at -20 °C. Dullbecco's modified eagle's medium (DMEM), fetal bovine serum, trypsin/EDTA, penicillin G/steptomycin antibiotics, 4' ,6-diamidino-2-phenylindole (DAPI) dihydrochloride solution, dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS) and ethanol were obtained from Beijing Solarbio Science and Technology Co., (Shanghai, China). Crystal violet stain (CV) (from: s.d.fine-CHEM Ltd), Coomassie blue R-250 (CB R-250), sodium dodecyl sulfate (SDS), formaldehyde, acetic acid (AA), and methanol were gifted from King Fahd Medical Research Center (KFMRC).
10
RNA Extraction and RT-qPCR Analysis
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The RNA from cell lines and tissues was extracted using Trizol (Shanghai Kangwei Biotech, China), chloroform, isopropanol, 75% ethanol (Solarbio, Beijing, China), and RNase-free water. The extracted RNA was quantified, followed by reverse transcription into cDNA using a reverse transcription kit (Servicebio, Wuhan, China). Subsequently, real-time fluorescence quantitative PCR was performed using 2 × SYBR Green PCR Master Mix (Servicebio, Wuhan, China). The relative expression levels of the target genes were calculated using the 2−ΔΔCt method. The experiments were repeated three times using different PCR runs, and the average values were obtained. β-actin was used as the internal reference gene.
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