Human MUC1 transgenic C57BL/6 mice were purchased from Dr. S. Gendler (Mayo Clinic, Scottsdale, AZ). IL-10−/− C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The MUC1+/− mice were crossed with IL-10−/− mice and then the F1 MUC1+/−/IL-10+/− mice were bred with IL-10−/− mice. Analysis of colon tissue was performed in the Mouse Histopathology Core, Dana-Farber/Harvard Cancer Center (R. T. Bronson, pathologist). Mucosa samples were disrupted into cell suspensions using the Tissue Tearor (BioSpec Products). These studies were performed under animal protocol number 12-029, approved by the Dana-Farber Institutional Animal Care and Use Committee (IACUC).
Tissue tearor
Manufactured by Biospec
Sourced in United States
The Tissue Tearor is a laboratory equipment used for the homogenization and disruption of tissues and cells. It features a high-speed motor that drives a sharp rotor, which tears and homogenizes the sample.
Automatically generated - may contain errors
Lab products found in correlation
Biopulverizer, by Biospec (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Multiskan go, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (5 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Enspire, by PerkinElmer (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Ultrasonic cell grinder, by Ningbo Xinzhi (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti β1 integrin, by Merck Group (2 mentions)
159 protocols using tissue tearor
1
MUC1 Transgenic and IL-10 Knockout Mice
2
Quantifying TREM-2 in Lung Tissue
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For lung tissue samples, lung tissue was minced and then homogenized in PBS containing protease inhibitor cocktail (cOmplete; Roche) using a rotor-type homogenizer (Tissue-Tearor; Biospec Products). For cell supernatant and lysate samples, BMDM (2 × 106 cells in 2 ml of complete DMEM containing 15% L cell–conditioned medium) were seeded into 12-well tissue plates, treated with or without IL-13 (20 ng/ml) or SeV (MOI 1 and 10). Cell supernatants were collected, and cells were washed twice with chilled PBS and then lysed in radio-immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) containing protease inhibitors. Levels of CSF-1 were determined using a Duoset ELISA kit (R&D Systems) and levels of cellular TREM-2 and sTREM-2 with a TREM-2 ELISA kit (Antibodies-online.com).
3
Cytokine Quantification in Cell Lysates
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After experimental treatments, cell membranes were disrupted and the cell lysates centrifuged at 10,000g at 4°C for 15 mins. The supernatant was separated from cell debris by a tissue tearor (Biospec, Bartlesville, OK, USA) and used for subsequent measurements. The concentrations of IL-1β, IL-6, IL-10 and TNF-a were measured using the corresponding ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.
4
Quantification of Hepatic Cyp2r1 Protein
5
Murine Organ Harvesting and Homogenization
6
Preparation of Human Amniotic Membrane Extracts
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Example 12
The entire procedure for preparation of total soluble human AM extracts (T) was carried out aseptically so as to be used for subsequent cell culture-based experiments. Frozen human placenta was obtained from Bio-Tissue, Inc. (Miami, Fla.), from which AM was retrieved. AM was sliced into small pieces to fit into the barrel of a BioPulverizer (Biospec Products, Inc., Bartlesville, Okla.), frozen in the liquid nitrogen, and then pulverized into a fine powder. The powder was weighed and mixed with cold PBS buffer (prepared by adding distilled H2O to 1×PBS, pH7.4, from 10×PBS, cat #70011-044, Invitrogen, Carlsbad, Calif.) with protease inhibitors (protease inhibitor cocktail, P8340, Sigma, and supplemented with 1 mM PMSF) and phosphatase inhibitors (50 mM sodium fluoride and 0.2 mM sodium vanadate) at 1:1 (ml/g). The mixture was kept on ice and homogenized with a Tissue Tearor (Biospec Products, Inc., Dremel, Wis.) for 5 times, 1 min each with a 2 min interval cooling. This water-soluble extract was designated as “Total” (T). The total water-soluble extract was mixed for 1 hr at 4° C., centrifuged at 4° C. for 30 min at 48000×g. The supernatant was divided into aliquots and stored at −80° C.
7
Western Blot Analysis of Tissue Lysates
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Lung and spleen tissues were homogenized with a tissue homogenizer (Tissue-Tearor, Biospec), and total lysate was obtained in 1% SDS, 1% IGEPAL, 0.5% NaDeoxycholate, 100 mM Hepes pH 8.0, following boiling for 10 minutes, 5 cycles of sonication for 30 seconds ON/ 30 seconds OFF and centrifugation at 14000 rpm for 20 minutes. The same procedure was applied to generate total cell lysate from LLC and EL4 tumor cells. Prior to Western blotting, protein concentrations were determined by BCA assay. 50 µg of proteins were separated by SDS-PAGE and transferred to PVDF membranes (#1620177, Bio-Rad). Blots were incubated for 1 hour at room temperature with anti-gelsolin, anti-STIP1, anti-GRP75, anti-HSP60, anti-PRDX6, and β-actin antibodies (See Table S1). Blots were incubated for 1 hour at room temperature with HRP-conjugated antibodies and developed with chemiluminescence reagents (SuperSignal West Pico Substrate, cat. #34580, ThermoFisher). All Western blots were scanned on FluoroChem M (ProteinSimple, San Jose, CA, USA), and band densitometry analysis was performed on all blots using Image J (NIH, Bethesda, MD). When multiple bands were detected around the expected size, they were all integrated in the calculation. All bands were normalized according to the background signals and then against β-actin. Once normalized, all experimental bands and lanes were compared with normal tissues.
8
Freshness Assessment of Oyster Meat
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The contents of malondialdehyde (MDA) and total sulfhydryl group (SH) of oyster meat at different storage days were determined to assess the freshness of oysters, i.e., to characterize the degree of lipid oxidation and protein structural integrity of oyster meat, respectively. Oyster meat with normal saline was first homogenized using Tissue-Tearor (BioSpec Products Inc., Bartlesville, OK, USA) at a ratio of weight (g): volume (mL) = 1:9, and the homogenate was centrifuged at 3000× g for 10 min at 4 °C to obtain supernatant. Then, the MDA and SH content in the supernatant of each sample were determined separately using commercial MDA and SH kits (purchased from Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), according to the manufacturer’s instructions [29 (link),30 (link)].
9
Extraction and Characterization of Sutherlandia frutescens
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Dried, milled vegetative parts of Sutherlandia frutescens (family: Fabaceae/Leguminosa), obtained from Big Tree Nutraceutical, Fish Hoek, South Africa were extracted in six different solvents: methanol, ethanol, acetone, acetonitrile, hot water, and cold water homogenization. Briefly, 1 g of dried SF was extracted in 50 ml of each solvent. Extraction in methanol, ethanol, acetone, and acetonitrile was done by adding respective solvent to the SF, followed by sonication for 20 min. Hot water extract was prepared by boiling SF for 20 min and cooling to room temperature. Cold water extract was prepared by homogenizing SF in water by tissue tearor (Biospec Products) for 20 min. All extracts were vacuum filtered and thereafter stored at 4°C until further use (20 mg/ml). For studies in cells, however, the hot water filtrate was freeze-dried for 72 h in the Savant refrigerated vapor trap (RVT4104-180) to obtain a dried powdered plant extract. Lyophilized extract was then dissolved in a serum-free media to a final concentration of 1 mg/ml stock solution, referred as a SFE (a yield of 5%).
10
Quantification of Inflammatory Mediators in Excised Paws
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