The dissected hippocampus was homogenized in lysis buffer mixed with a protease inhibitor cocktail (Sigma–Aldrich Corp., MI, USA) utilizing Tissue Master-125 homogenizer (Omni International, Kennesaw, GA, USA) (Al-Sawalha, et al. 2017 ). The levels of thiobarbituric acid reactive substances (TBARS) (Cayman Chemical, MI, USA) and brain derived neurotrophic factor (BDNF) (R&D Systems, MN, USA) were measured by ELISA kits following the manufacturers’ instructions. The activity of anti-oxidant enzymes; catalase (Cayman Chemical, MI, USA), glutathione peroxidase (GPx) (Sigma–Aldrich Corp., MI, USA) and superoxide dismutase (SOD) (Sigma–Aldrich Corp., MI, USA) were measured according to the manufacturers’ instructions. The optical density was measured at the specified wavelengths as per kit’s manual using an Epoch Biotek microplate reader (BioTek, Winooski, VT, USA). The concentration of total protein was determined by a commercially available kit (BioRAD, Hercules, CA, USA). The levels of TBAR, BDNF, VGEF and activities of the anti-oxidant enzymes were normalized to total protein in each sample.
Tissue master 125 homogenizer
Manufactured by Omni International
Sourced in United States
The Tissue Master 125 is a homogenizer designed for the rapid and efficient disruption of tissue samples. It operates using a high-speed rotary blade to thoroughly homogenize the sample, ensuring complete disruption of the tissue structure.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Glutathione peroxidase gpx, by Merck Group (1 mentions)
Epoch biotek microplate reader, by Agilent Technologies (1 mentions)
Brain derived neurotrophic factor (bdnf), by R&D Systems (1 mentions)
Catalase, by Cayman Chemical (1 mentions)
Tbars, by Cayman Chemical (1 mentions)
Superoxide dismutase sod, by Merck Group (1 mentions)
Zetasizer, by Malvern Panalytical (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qiashredder, by Qiagen (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
7 protocols using tissue master 125 homogenizer
1
Oxidative Stress and BDNF Measurement
2
Formulation and Characterization of Polyphenol-Loaded Nanoemulsions
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The solubility of polyphenon 60 and cranberry were checked in different oils, surfactants and co-surfactants. The nanoemulsion was prepared by dissolving cranberry in oleic acid as oil phase, tween 20 as surfactant and glycerol as a co-surfactant. P60 was dissolved in milli-Q water to prepare aqueous phase and added drop wise to the oil phase with continuous stirring to form pre emulsion. This pre-emulsion was subjected to high shear homogenization using Tissue Master 125 homogenizer (Omni International, Georgia) at 10,000 rpm for 20 min under ice bath and further subjected to high energy ultra-sonication via Bench Top Ultrasonicator (Model UP400S, 24 KHz 400 W, Hielcher, Ultrasound Technology, Germany) at amplitude of 70% for 286 sec at 0.3s ON and 0.7s OFF cycles. The prepared nanoemulsion was characterized for particle size and zeta potential determination using Malvern Zetasizer (Malvern, Worcestershire, UK). Before determining particle size and zeta potential, the nanoemulsion was diluted with HPLC water at ratio of 1:50 v/v.
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The solubility of polyphenon 60 and cranberry were checked in different oils, surfactants and co-surfactants. The nanoemulsion was prepared by dissolving cranberry in oleic acid as oil phase, tween 20 as surfactant and glycerol as a co-surfactant. P60 was dissolved in milli-Q water to prepare aqueous phase and added drop wise to the oil phase with continuous stirring to form pre emulsion. This pre-emulsion was subjected to high shear homogenization using Tissue Master 125 homogenizer (Omni International, Georgia) at 10,000 rpm for 20 min under ice bath and further subjected to high energy ultra-sonication via Bench Top Ultrasonicator (Model UP400S, 24 KHz 400 W, Hielcher, Ultrasound Technology, Germany) at amplitude of 70% for 286 sec at 0.3s ON and 0.7s OFF cycles. The prepared nanoemulsion was characterized for particle size and zeta potential determination using Malvern Zetasizer (Malvern, Worcestershire, UK). Before determining particle size and zeta potential, the nanoemulsion was diluted with HPLC water at ratio of 1:50 v/v.
3
Traumatic brain injury biomarker analysis
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Animals were sacrificed by decapitation under isoflurane anaesthesia at t = 4 hours, 24 hours, and 7 days after trauma or sham surgery. Whole blood was taken by intracardiac puncture immediately prior to decapitation. For collection of serum samples, whole blood was transferred to sterile serum microtubes (Sarstedt, Nümbrecht, Germany), centrifuged at 10,000 g for 10 minutes (4°C) and the supernatants were frozen at -80°C until analysis. For collection of plasma samples, whole blood was collected in sterile microtubes containing 3.5% ethylenediaminetetraacetic acid (EDTA) and a protease inhibitor cocktail (Sigma-Aldrich, St Louis, Missouri, United States). Samples were centrifuged at 2,000 g for 15 minutes (4°C) and the obtained plasma stored at -80°C until analysis. Brain samples were surgically removed after decapitation, snap-frozen in liquid nitrogen and stored at -80°C. Prior to analysis, brains were split into left (injured) and right (uninjured internal control) hemispheres, and the cerebellum was removed. For assessment of inflammatory and apoptotic mediators by western blots, brain hemispheres were homogenized with a Tissue Master-125 Homogenizer (Omni International, Kennesaw, Georgia, United States), as described in detail below.
4
RNA Extraction and Sequencing Protocol
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Tissue was homogenized using the Tissue Master 125 homogenizer (OMNI International) and/or QIAshredder (Qiagen). RNA was extracted with RNeasy mini kit (Qiagen) following the supplier's instructions. For measurement of RNA quantity, ND1000 Spectrophotometer (NanoDrop Technologies) and/or Agilent 2100 Bioanalyzer was used. RNA-seq libraries were processed and sequenced on a HiSeq V4 (SR 50) by the DKFZ Genomics Core Facility.
5
Quantifying Antibiotic-Resistant E. coli
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Fecal samples were collected at the indicated times, diluted in sterile PBS, and then plated on LB agar containing kanamycin (50 μg/ml) and either rifampicin (50 μg/ml) or nalidixic acid (50 μg/ml) to quantify the number of E. coli mcr-1+ and E. coli mcr-1-. Bacterial identification was performed using chromogenic medium or mass spectrometry, and the presence of mcr-1 in isolates was confirmed by PCR. No such resistant or mcr-1-positive bacteria were found in uninfected mice. Intestinal tissues were longitudinally opened, washed in 3 ml of sterile PBS, and then either homogenized using a Tissue Master 125 Homogenizer (Omni International) to quantify E. coli tissue-associated loads or directly incubated in sterile DMEM containing antibiotics to quantify the secreted cytokines.
6
Protein Extraction and Quantification
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Protein was extracted from tissue or cells by addition of RIPA buffer (Sigma Aldrich) including complete protease inhibitor cocktail tablets (Roche) according to the manufacturer's instruction. Tissue was homogenized using the Tissue Master 125 homogenizer (OMNI International) or sonication after adding lysis buffer directly to the frozen sample. Cell lysates were vortexed three to four times every 10 min for a total of 30 min and were kept on ice in between. Lysates were centrifuged at 17,000g for 30 min at 4°C. Protein concentration of cell lysates was measured using Pierce BCA protein assay (Thermo Fisher Scientific), following the supplier's instructions. Primary antibodies are listed in Supplemental Table S5 .
7
Quantifying Bacterial Shedding and Colonization in Murine Intestine
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Fresh stool samples were collected from infected mice 1, 2, 3, and 4days post-LF82 challenge to determine bacterial fecal shedding. Fecal pellets standardized to a concentration of 100mg ml−1 in PBS were homogenized and serial 10-fold dilutions performed. About 10μl of diluted samples was plated on Eosin Methylene Blue (EMB) differential medium agar containing ampicillin (100μg ml−1) and erythromycin (500μg ml−1) and incubated at 37°C overnight. Ileum, caecum, and colon samples were collected 4days post-infection in cold PBS at necropsy and homogenized using Tissue Master 125 Homogenizer (OMNI International). Homogenate tissues were serially diluted and plated on EMB agar containing ampicillin (100μg ml−1) and erythromycin (500μg ml−1). Colonies were counted after 24–48h of incubation at 37°C and expressed as c.f.u. per gram of tissue.
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