Total RNA was extracted separately from 32 paired CRC tissues, surgical margin tissues, and five CRC cell lines using TRIzol® reagent (Life Technologies, Carlsbad, CA, USA). Genomic DNA was separately obtained from 24 CRC tissues, six normal colorectal tissues, and the five CRC cell lines using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The concentration of DNA and RNA were measured using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Rockford, IL, USA) and stored at −80°C. The experimental and control group of the HT-29 and SW480 cells were lysed using a protein extraction reagent (Thermo Scientific) that contained the protease inhibitor, phenylmethane sulfonyl fluoride, and a phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and the lysate was then homogenized using a Ultrasonic Cell Grinder (Scientz, Ningbo). The supernatant was collected after centrifugation, and the concentration of protein in the supernatant was determined using the BCA protein kit (Thermo Scientific).
Ultrasonic cell grinder
Manufactured by Ningbo Scientz Biotechnology
Sourced in United States, China
The Ultrasonic cell grinder is a laboratory instrument designed to disrupt cells and release their contents through the application of high-frequency sound waves. It is used to prepare samples for various analyses and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Protein extraction reagent, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Bca protein kit, by Thermo Fisher Scientific (4 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
0.2 μm filter, by Pall Corporation (1 mentions)
Malonaldehyde, by Nanjing Jiancheng (1 mentions)
6 protocols using ultrasonic cell grinder
1
Comprehensive Biomolecular Extraction from Colorectal Tissues
2
RNA and Protein Extraction from Cell Lines
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Total RNA was extracted from tissues and cell lines using TRIzol® reagent (LifeTechnologies, Carlsbad, CA, USA). Genomic DNA was isolated from tissues and cell lines using the QIAamp DNAMini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. The DNA and RNA were stored at −80 °C after measuring their concentrations of them using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Rockford, IL, USA).The experimental and control groups of the HCT116 and LoVo cells were lysed using a protein extraction reagent (Thermo Scientific) that contained the protease inhibitors, phenylmethane sulfonyl fluoride, and a phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and the lysate was then homogenized using an Ultrasonic Cell Grinder (Scientz, Ningbo). The supernatant was collected after centrifugation, and protein concentration of protein in the supernatant was determined using the BCA protein kit (Thermo Scientific).12 (link)
3
Optimizing BoHV-1 Virus Yield in MDBK Cells
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BoHV-1 was passaged 3 times in suspended MDBK cells, quantitated and designated as the seed virus. Suspension-adapted MDBK cells at the densities of 2.0 × 106 cells/mL, 4.0 × 106 cells/mL and 8.0 × 106 cells/mL were infected with the seed virus at the multiplicity of infection (MOI) of 0.1, 0.05 or 0.01. Cells were then harvested with medium at different time points post infection, frozen and thawed 3 times, and the supernatant was stored for virus titration with the protocol described above.
Furthermore, the following methods for virus release were compared for their effects on the recovery of viruses from the MDBK cells. (1) Repeated freeze–thaw cycles (freezing at −80 °C, thawing at room temperature). (2) Ultrasonic treatment. The sonication was performed with the ultrasonic cell grinder (Scientz, Ningbo, China) and the output power was 180 W. The working volume was 0.5 to 1.0 mL, and the infected culture was treated for 1 to 10 min. Additionally, the treated virus suspension was then centrifuged at 3000 rpm for 5 min and the supernatant was filtered by 0.2 μm filter (Pall Corporation, Port Washington, NY, USA) for virus titer determination.
Furthermore, the following methods for virus release were compared for their effects on the recovery of viruses from the MDBK cells. (1) Repeated freeze–thaw cycles (freezing at −80 °C, thawing at room temperature). (2) Ultrasonic treatment. The sonication was performed with the ultrasonic cell grinder (Scientz, Ningbo, China) and the output power was 180 W. The working volume was 0.5 to 1.0 mL, and the infected culture was treated for 1 to 10 min. Additionally, the treated virus suspension was then centrifuged at 3000 rpm for 5 min and the supernatant was filtered by 0.2 μm filter (Pall Corporation, Port Washington, NY, USA) for virus titer determination.
4
Cellular Antioxidant Evaluation Protocol
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At the end of treatment, the cells were scraped off using cell scrapers, and the supernatant was discarded after centrifugation at 1000× g for 10 min at room temperature. Next, 1 mL PBS was added to the cell precipitate for washing and the supernatant was centrifuged at 1000× g for 10 min to leave the cell precipitate. Then, the cell precipitate was washed twice. A total of 400 µL PBS was added to the cell precipitate and homogenized by blowing using a pipettor. The cells were crushed in an ice-water bath using an ultrasonic cell grinder (Scientz, Ningbo, China) with a power of 300 W, 3 s each sonication, 30 s intervals, and 5 repetitions. In the end, the supernatant was centrifuged at 12,000× g for 10 min at 4 °C, and the malonaldehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT) were detected according to the instructions of commercial kits (Jiancheng, Nanjing, China). All reactions were repeated three times (n = 3).
5
Extraction and Quantification of RNA and Protein from Colorectal Cancer Cells
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Total RNA was extracted from CRC tissues and cell lines with TRIzol® reagent (Life Technologies, Carlsbad, CA, USA). The extracted RNA was measured by NanoDrop 2000 spectrophotometer (Thermo Scienti c, Rockford, IL, USA) and stored at -80 °C. Total protein was extracted from HCT116, RKO and HT-29 cells. The cells were lysed by protein extraction reagent (Thermo Scienti c) in the experimental group and the control group, then the lysate was collected into EP tubes and homogenized by ultrasonic cell grinder (Scientz, Ningbo). After centrifugation at 4°C, the supernatant was transferred to new EP tubes. The protein concentration was measured by BCA protein kit (Thermo Scienti c) then stored in -80 °C refrigerator [6] .
6
Extraction and Quantification of RNA and Protein from Colorectal Cancer Cells
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Total RNA was extracted from CRC tissues and cell lines with TRIzol® reagent (Life Technologies, Carlsbad, CA, USA). The extracted RNA was measured by NanoDrop 2000 spectrophotometer (Thermo Scienti c, Rockford, IL, USA) and stored at -80 °C. Total protein was extracted from HCT116, RKO and HT-29 cells. The cells were lysed by protein extraction reagent (Thermo Scienti c) in the experimental group and the control group, then the lysate was collected into EP tubes and homogenized by ultrasonic cell grinder (Scientz, Ningbo). After centrifugation at 4°C, the supernatant was transferred to new EP tubes. The protein concentration was measured by BCA protein kit (Thermo Scienti c) then stored in -80 °C refrigerator [6] .
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