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7 protocols using bullet blender gold homogenizer

1

Ectoparasites Identification and Bartonella Detection

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We collected ectoparasites from the skin and pelage of bats and stored them in microcentrifuge tubes with 70% ethanol. Ectoparasite species were identified by using available morphologic keys (17 (link)), and identifications were later confirmed by sequencing of the mitochondrial 16S rRNA and cytochrome oxidase I (COI) genes (18 ,19 (link)).
Using a Bullet Blender Gold homogenizer (Next Advance, Averill Park, NY, USA), we homogenized whole ectoparasites in Navy Eppendorf bead tubes (Next Advance) containing 400 μL brain–heart infusion broth (CDC, Atlanta, GA, USA). We extracted DNA from the homogenates by using the KingFisher Flex Purification System and the associated MagMAX Pathogen RNA/DNA Kit (both ThermoFisher, Waltham, MA, USA) according to the manufacturer’s protocols. Detection of Bartonella DNA in ectoparasite samples was performed by nested PCR for gltA (20 (link)) because of low concentrations of DNA and by conventional PCR for ITS (21 (link)), followed by sequencing and sequence analysis of amplicons.
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2

Tissue Analysis of Murine Placenta

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At the time of sacrifice (gd12.5), implantation sites were removed and decidua, placenta and fetus were carefully dissected; tissue weights were recorded prior to storage. Individual tissues were placed in 500 µL PBS in sterile RINO® tubes (TUBE1R5-S; FroggaBio, Toronto, ON, Canada) with a mixture of small and large stainless-steel beads. Tissues were homogenized for 5 min at 4 ℃ at maximum speed using a Bullet Blender® Gold Homogenizer (NextAdvance, Troy, NY, USA). Following homogenization, samples were centrifuged at 8000 rpm for 5 min. Supernatants were collected and stored at −80 ℃ until future use. For cytokine/chemokine analysis, 80 µL of undiluted tissue homogenate supernatant was sent to Eve Technologies (Calgary, AB, Canada) for quantification using a mouse 31-plex array (MD31; Eve Technologies). Cytokine/chemokine quantification was performed using 2 implantation sites from 3 pregnant gd12.5 mice in normal, low dose (103 pfu/mL) and high dose (105 pfu/mL) groups.
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3

Bartonella Isolation from Rodent Hearts

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Rodent hearts (∼50 mg) were homogenized in 200 μL brain heart infusion broth supplemented with amphotericin B (final concentration 25 μg/mL) using Bullet Blender® Gold homogenizer (Next Advance Inc., New York, NY) following heart protocol provided by the manufacturer. The homogenates (100 μL) were then plated on heart infusion agar supplemented with 10% rabbit blood and incubated in an aerobic condition with 5% CO2 at 35°C for up to 4 weeks. Plates were monitored for bacterial growth twice a week after initial plating. Bacterial colonies were presumptively identified as probable Bartonella species based on their morphological characteristics. A single colony from each plate was picked and streaked onto secondary agar plates for another 4–5 days of incubation under the same condition. If colonies on the same plate looked different, a single colony for each appearance is subcultured separately. Pure cultures were harvested in 10% glycerol.
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4

Measuring Heat Stress Protein Synthesis

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Cells were subjected to HS at 40°C for 1 h. Samples were then serially diluted (10x) and 10 μL aliquots were spotted onto YES plates and incubated at 30°C for 1 to 4 days. To determine the growth rates of the different strains after HS, liquid cultures were allowed to recover at 30°C and samples were taken at various time intervals for OD600nm absorbance spectroscopic measurements (WPA CO8000, Biochrom Ltd., Cambridge, UK). In choline supplemenation experiments, 1 mM choline was added to the culture during HS.
De novo protein synthesis was measured by adding 20 μL of 14C protein hydrolysate (Amersham CFB25, 50 μCi/mL) to 2 mL cell suspension for 1 h before lysis. After brief centrifugation, cells were resuspended in 250 mM NaOH and disrupted using a Bullet Blender Gold homogenizer (Next Advance, Inc., Averill Park, NY, USA) in the presence of zirconium oxide beads (0.5 mm) at speed 8 for 3 min at 4°C. The homogenate was centrifuged (1000 xg, 4°C, 3 min) and proteins were precipitated on ice by mixing samples with an additional 1/4 sample volume of 50% (v/v) aqueous TCA for 20 min. After centrifugation (10,000 xg, 4°C, 10 min), pellets were washed twice with 1 mL −20°C acetone and solubilized in 1x protein loading buffer for 3 h. Proteins were separated on 10% SDS-PAGE and prepared for fluorography as in [44 (link)].
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5

Muscle RNA Extraction and RT-qPCR Analysis

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Muscles were flash frozen in Trizol (Ambion) at dissection and homogenized using a Bullet Blender Gold homogenizer (Next Advance BB24‐AU). RNA was isolated using an RNeasy Plus Mini Kit (Qiagen) following the manufacturer's instructions. cDNA was synthesized using 500 or 1000 ng of RNA (from EDL/SOL or gastrocnemius, respectively) using qScript cDNA SuperMix (QuantaBio). Reverse transcription‐quantitative PCR (RT‐qPCR) was performed on a Step One Plus Real‐Time PCR machine (Applied Biosystems) using PerfeCTa SYBR Green FastMix (QuantaBio). All reactions utilized the following thermal cycler conditions: 50°C for 2 min, 95°C for 2 min, 40 cycles of a two‐step reaction, denaturation at 95°C for 15 s, and annealing at 60°C for 30 s. Experiments were standardized to YWAZ. Primers are provided in TableS1.
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6

Liver Biopsy Sampling for Bariatric Research

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Liver wedge biopsies were obtained from individuals enrolled in the Bariatric Surgery Program at the Geisinger Clinic Center for Nutrition and Weight Management [47 ]. Details of the study population can be found elsewhere [[48] , [49] (link), [50] (link)]. All study participants provided written informed consent for research, which was conducted according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). The Institutional Review Boards of Geisinger Health System, Translational Genomics Research Institute, and Temple University School of Medicine approved the research protocol. Liver tissue was cut into ~5–10 mg pieces and added to sterile microcentrifuge tubes (Next Advance; Troy, NY) with RLT Plus lysis buffer containing β-mercaptoethanol (Qiagen), and homogenized in a Bullet Blender Gold homogenizer (Next Advance) for 12–15 min. Total RNA was extracted from homogenized lysate using the RNeasy Mini Kit (Qiagen) and quantified by the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Quantitative PCR was performed as described above.
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7

Quantitative Trehalose Analysis in Cell Lysates

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For cell lysate preparation, 108 cells were harvested by filtration, resuspended in 300 μL ice-cold water, and disrupted using a Bullet Blender Gold homogenizer (Next Advance, Inc., Averill Park, NY, USA), in the presence of zirconium oxide beads (0.5 mm), at speed 8 for 3 min at 4 °C. For trehalose quantitation, the lysate was boiled for 5 min, centrifuged at 10,000× g for 5 min, and a 5 μL aliquot was digested in 45 μL 135 mM citrate buffer (pH 5.6) in the presence of 1.15 mU trehalase (Merck, Darmstadt, Germany) at 37 °C overnight. Glucose content was determined by adding 100 μL of Assay Reagent (GO Assay kit, Merck, Darmstadt, Germany) and incubated at 37 °C for 30 min. Reactions were stopped by the addition of 100 μL 12 N sulfuric acid, and the absorbance was determined by Multiskan EX plate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 560 nm. trehalose and glucose solutions (25–100 μg/mL) were used as standards. Protein level of the lysates was measured using the Micro BCATM Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
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