Isolated brains from Sod1−/− or Sod1+/+ mice were frozen and stored at −70°C. Prior to tissue lysate preparation, brains were equilibrated to −20°C for 30 min, minced into small pieces and 1 g tissue was resuspended in 10 mL ice-cold T-PER Tissue Protein Extraction Reagent with 200 μM NAM, 10 μM TSA, 10 μM Tubastatin A and HALTTM protease and phosphatase inhibitors. Following a 30-min incubation on ice, tissues underwent five cycles of sonication on ice (20 s at output 5 and 50% duty cycle) using a Branson Sonifer 450. Tissue lysates were then passed through a syringe fitted with a 25 1/2 gauge needle until no longer viscous. Cellular debris was removed by centrifugation at 18,200× g for 30 min at 4°C in an Eppendorf 5417R microcentrifuge. Supernatants were collected and protein concentrations were determined using a Pierce Bradford assay kit.
Sonifer 450
Manufactured by Emerson
The Sonifer 450 is a laboratory equipment product manufactured by Emerson. It is a device used for the purpose of sonication, a process that utilizes high-frequency sound waves to disrupt and homogenize samples. The core function of the Sonifer 450 is to provide controlled and consistent sonication to facilitate various laboratory procedures and experiments.
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Lab products found in correlation
12 protocols using sonifer 450
1
Sod1 Deficient Mouse Brain Protein Extraction
2
Characterization of Cellulose Nanocrystals and Asbestos
3
Preparation of SARM1 SAM-TIR Protein from Transfected HEK293T Cells
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NRK1-HEK293T cells (Essuman et al., 2017 (link)) were seeded onto 150 cm2 flasks at 10 × 106 cells per plate. The next day, the cells were transfected with 15 μg of human SAM-TIR expression plasmid using X-TremeGENE 9 DNA Transfection Reagent. Human SAM-TIR expression plasmid consisted of a Strep-TEV-human SARM1, aminoacids 408–700, cloned in pSF-CMV-Amp using NcoI and XbaI sites. The cultures were supplemented with 1 mM nicotinamide riboside (NR) at time of transfection to minimize toxicity from SAM-TIR overexpression (Essuman et al., 2017 (link)). Forty-eight hours after transfection, cells were harvested, pelleted by centrifugation at 1,000 rpm (Sorvall ST 16R centrifuge, ThermoFisher), and washed once with cold PBS (0.01 M phosphate buffered saline NaCl 0.138 M; KCl 0.0027 M; pH 7.4). The cells were resuspended in PBS with protease inhibitors (cOmpleteTM protease inhibitor cocktail) and cell lysates were prepared by sonication (Branson Sonifer 450, output = 3, 20 episodes of stroke). The lysates were centrifuged (12,000 × g for 10 min at 4°C) to remove cell debris and the supernatants (containing SARM1 SAM-TIR protein) were stored at −80°C for later use in the in vitro SARM1 SAM-TIR NADase assay (see below). Protein concentration was determined by the Bicinchoninic (BCA) method and used to normalize lysate concentrations.
4
Fabrication of ICG Nanobubbles
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Briefly, 100 mg of ICG was dissolved in 1 mL of PVA solution (1 w/v%). Two hundred microliter of the ICG-PVA solution was added in drops to 10 mL of PLGA/dichloromethane (0.25 w/v %) in an ice-water bath under sonication to form the first emulsion. Here, the PLGA polymer contains the mixture of 80 wt% of PLGA, 10 wt% of poly(lactide-co-glycolide)-b-poly(ethylene glycol) (PLGA-PEG), and 10 wt% of poly(lactide-co-glycolide)-b-poly(ethylene glycol)-maleimide (PLGA-PEG-MAL) copolymer. Typically, for nanobubble around 250 nm in diameter, the sonicator (Branson Sonifer 450) was set in a pulse width of 5 s on and 5 s off period, the sonication power was 20% of total power, and the sonication period was 5 min in total. The first emulsion was transferred and added dropwise to 10 mL of PVA solution (1 w/v %) under sonication (5 s on-off in 20% power for 5 min) in an ice-water bath to form second emulsions. The second emulsion was poured slowly to 20 mL of isopropanol (5% v/v) in DI water solution and stirred for more than 2 h with magnetic stirring. The ICG nanoparticles were centrifuged at 1,500 rpm for 20 min, re-suspend with water and washed twice with a centrifuge. The ICG nanoparticles were then lyophilized for 48 h to form ICG nanobubbles.
5
Protein Extraction from C. elegans
6
Overexpression and Purification of SARM1 SAM-TIR Domain
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NRK1-HEK293T cells3 (link) were seeded onto 150-cm2 flasks at 10 × 106 cells per plate. The next day, the cells were transfected with 15 μg of human SAM-TIR expression plasmid using X-TremeGENETM 9 DNA Transfection Reagent. The human SAM-TIR expression plasmid consisted of Strep-TEV-human SARM1, amino acids 408–700, cloned into pSF-CMV-Amp using NcoI and XbaI sites. The cultures were supplemented with 1 mM nicotinamide riboside at the time of transfection to minimize toxicity from SAM-TIR overexpression.3 (link) Forty-eight hours after transfection, cells were harvested, pelleted by centrifugation at 1000 rpm (Sorvall ST 16 R centrifuge, ThermoFisher) and washed once with cold PBS (0.01 M PBS; NaCl 0.138 M; KCl 0.0027 M; pH 7.4). The cells were resuspended in PBS with protease inhibitors (cOmpleteTM protease inhibitor cocktail) and cell lysates were prepared by sonication (Branson Sonifer 450, output = 3, 20 episodes of stroke). The lysates were centrifuged (12 000g for 10 min at 4°C) to remove cell debris and the supernatants (containing SARM1 SAM-TIR protein) were stored at −80°C for later use in the in vitro SARM1 SAM-TIR NADase assay. Protein concentration was determined by the bicinchoninic (BCA) method and used to normalize lysate concentrations.
7
PEG10 Protein Expression Analysis
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Cells were trypsinized and collected by centrifugation (130× g, 8 min, room temperature) 24 h after transfection. The pellet was resuspended in 500 μL of phosphate-buffered saline containing protease inhibitor cocktail (complete EDTA-free Protease Inhibitor Tablet, ROCHE, St. Louis, MO, USA). The pellets were lysed by sonication (Branson Sonifer 450, duty cycle 30 %, output control 4) for 3 × 10 s. Cell lysates were then centrifuged at 16,000× g for 15 min at 4 °C to remove cellular debris. For the comparison of expressed PEG10 proteins, 30 μL aliquots of the supernatants were analyzed by Western blot.
8
Protein Extraction from HEK293 Cells
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HEK293 cells seeded onto 145 mm culture dishes in 20 mL growth medium were grown to near confluency and treated with deacetylase inhibitors (10 mM NAM plus 1 μM TSA) or a DMSO vehicle control. After a 6-h treatment at 5% CO2 and 37°C, cells were harvested as described in 2.5 and resuspended in ice-cold Tissue Protein Extraction Reagent (T-PER) supplemented with 200 μM NAM, 10 μM TSA and HALTTM protease and phosphatase inhibitors at a volume of 10 mL/g cell pellet. Cells were then subjected to three freeze/thaw cycles using a dry ice/ethanol bath and 37°C water bath followed by one cycle of sonication on ice (30 s at output 5 and 50% duty cycle) using a Branson Sonifer 450. Cellular debris was removed by centrifugation at 18,200× g for 20 min at 4°C in an Eppendorf 5417R microcentrifuge. Cleared supernatants were saved and protein concentrations were determined with a Pierce Bradford protein assay kit.
9
Preparation of CNT/F Aqueous Suspensions
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Aqueous stock suspensions of CNT/F were generated by weighing the dry powder and suspending in well-characterized dispersion medium [DM; 0.6 mg/ml mouse serum albumin + 0.01 mg/ml 1,2-dipalmitoyl-sn-glycero-3-phosphotidyl (DPPC) in phosphate-buffered saline (PBS) without calcium and magnesium] [132 (link)] at 2 mg/ml concentration. The stock suspension was sonicated for 5 min at 70% amplitude using a cup horn sonicator (Sonics VibraCell VCX-750 with Cup-type Sonicator; Newton, CT) immersed in continuous flowing cold water. The samples were vortexed intermittently after every minute for 10 s. The stock solution at 2 mg/ml was dispersed in cell culture media by diluting to highest test concentration i.e. 24 μg/ml. The CNT/F containing cell culture media was then subjected to probe tip sonication (Branson Sonifer 450, continuous output) for a total of 2 min, with 10 s vertexing after every 30 s. CNT/F containing cell culture media at 0.024, 0.24 or 2.4 μg/ml were obtained by serial dilution.
10
Oral administration of MAP4343 and fluoxetine in tree shrews
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MAP4343 was dissolved in 0.5% hydroxyethylcellulose by sonication during 3 cycles of 15 pulses separated by 15 seconds on ice (Branson Sonifer-450). Animals received per os administration of MAP4343 (50mg/kg/d), fluoxetine (15mg/kg/d; Fluoxetin ratiopharm Lösung, Ratiopharm, Ulm, Germany), or the vehicle (hydroxyethylcellulose) each day during the treatment period (Figure 1A ). Drugs were administrated between 8:00 and 8:30 am . A detailed methodology for oral pharmacological treatment in tree shrews was described by Schmelting et al., 2014 (link).
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