Anopheles gambiae, An. stephensi, An. albimanus, An. freeborni, An. dirus, Culex quinquefasciatus, Lutozomyia longipalpis, and Aedes aegypti were reared at the NIAID/Laboratory of Malaria and Vector Research mosquito facility under the supervision of Mr. André Laughinhouse. Salivary glands were dissected under the microscope, placed in PBS (30 pairs/30 µl) and frozen at −80°C. When required, the tubes were sonicated in a Misonix 3000 sonicator (Farmingdale, NY) and centrifuged for 5 min at 13,000×g. The supernatant was collected (herein named salivary gland homogenate, SGH) and frozen at −80°C.
Misonix sonicator 3000
Manufactured by Bioventus
Sourced in United States
The Misonix Sonicator 3000 is a laboratory equipment used for sonication, a process that utilizes high-frequency sound waves to disrupt the cell membranes of biological samples. The device generates and delivers ultrasonic energy to facilitate the extraction, homogenization, or cell lysis of various materials.
Automatically generated - may contain errors
Lab products found in correlation
Salmon sperm dna protein a agarose beads, by Merck Group (2 mentions)
Millipore chip kit, by Merck Group (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Complete protease inhibitor tablet, by Roche (1 mentions)
Amplex red cholesterol assay kit, by Thermo Fisher Scientific (1 mentions)
Millex gp filter, by Merck Group (1 mentions)
Protease inhibitors cocktail, by Merck Group (1 mentions)
Protein g beads, by Merck Group (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Sonicated salmon sperm dna, by Merck Group (1 mentions)
Ab7970, by Abcam (1 mentions)
11 protocols using misonix sonicator 3000
1
Mosquito Salivary Gland Extraction Protocol
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Anopheles gambiae, An. stephensi, An. albimanus, An. freeborni, An. dirus, Culex quinquefasciatus, Lutozomyia longipalpis, and Aedes aegypti were reared at the NIAID/Laboratory of Malaria and Vector Research mosquito facility under the supervision of Mr. André Laughinhouse. Salivary glands were dissected under the microscope, placed in PBS (30 pairs/30 µl) and frozen at −80°C. When required, the tubes were sonicated in a Misonix 3000 sonicator (Farmingdale, NY) and centrifuged for 5 min at 13,000×g. The supernatant was collected (herein named salivary gland homogenate, SGH) and frozen at −80°C.
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Anopheles gambiae, An. stephensi, An. albimanus, An. freeborni, An. dirus, Culex quinquefasciatus, Lutozomyia longipalpis, and Aedes aegypti were reared at the NIAID/Laboratory of Malaria and Vector Research mosquito facility under the supervision of Mr. André Laughinhouse. Salivary glands were dissected under the microscope, placed in PBS (30 pairs/30 µl) and frozen at −80°C. When required, the tubes were sonicated in a Misonix 3000 sonicator (Farmingdale, NY) and centrifuged for 5 min at 13,000×g. The supernatant was collected (herein named salivary gland homogenate, SGH) and frozen at −80°C.
2
Quantification of Prion Amplification
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Protein misfolding cyclic amplification was performed as previously described [57 (link)]. Briefly, 10% w/v brain homogenate (500 μg eq.) was 2-fold serially diluted in DPBS. Diluted samples were further diluted 1:20 into uninfected Syrian hamster brain homogenate in PMCA conversion buffer (phosphate-buffered saline [pH 7.4] containing 1 mM ethylenediaminetetraacetic acid [EDTA; Sigma-Aldrich, St. Louis, MO],1% [v/v] Triton X-100 [Sigma-Aldrich, St. Louis, MO], and complete protease inhibitor tablet [Roche Diagnostics, Mannheim, Germany]) and four 100 μl aliquots made per dilution (three replicates, one frozen, unsonicated control). Samples were loaded into a Misonix 3000 sonicator (Farmingdale, NY) and subjected to one round of PMCA (cycles of 5 second sonication, 9 minute 55 second incubation for 24 hours). Following PMCA, PrPSc was detected and quantified via Western blot as described above. For both protocols, the PMCA conversion coefficient is calculated as the reciprocal of the concentration of the highest dilution of prion-infected brain homogenate that resulted in detectable amplified PrPSc by Western blot following one round of PMCA.
3
Micellar Cholesterol Solubility Assay
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Micellar cholesterol solubility was measured after adding 3 mg/mL of each peptide fraction to a suspension of intestinal micelles prepared in vitro. Cholesterol micelles were prepared according to the method of Zhang et al. (2012) (link) with some modifications. Sodium phosphate buffer at pH 7.4 containing 6.6 mmol/L taurocholate and 132 mmol/L NaCl salts was mixed with 2 mmol/L cholesterol and 1 mmol/L linoleic acid. To form the micelles, the suspension was sonicated twice for 1 min using a Misonix 3000 sonicator (Misonix, New York, NY) at 95% energy output (114 W). The supernatant fractions were filtered through a 0.22-μm Millex-GP filter (Millipore, Bedford, MA). The supernatant fractions (25 μL) were collected, and cholesterol concentrations were determined using an Amplex Red Cholesterol Assay Kit (Thermo Fisher Scientific, Waltham, MA) by measuring the absorbance at 510 nm. Cholesterol concentrations were determined from standard curves using cholesterol calibration standards. Cholesterol concentrations and inhibition ability were calculated using the following equations:
where OD c is the calibrator's absorbance measured by optical density and OD s is the sample's absorbance; and
where C o is the cholesterol concentration of the original micelles and C s is the cholesterol concentration after the peptide fraction was added (Zhang et al., 2012; (link)Marques et al., 2015a) .
where OD c is the calibrator's absorbance measured by optical density and OD s is the sample's absorbance; and
where C o is the cholesterol concentration of the original micelles and C s is the cholesterol concentration after the peptide fraction was added (Zhang et al., 2012; (link)Marques et al., 2015a) .
4
Prion Protein Amplification by PMCA
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Protein misfolding cyclic amplification (PMCA) was performed as previously described (71 (link)). Samples (n ≥ 3) in PMCA conversion buffer were placed into polypropylene tubes in a Misonix 3000 sonicator (Misonix, Farmingdale, NY). The average output of the sonicator was 165 W during each sonication cycle. A PMCA round consisted of 144 cycles of a 5-s sonication, followed by an incubation of 9 min 55 s at 37°C. After each round of PMCA, an aliquot of sonicated sample was added to fresh 10% (wt/vol) uninfected brain homogenate in PMCA conversion buffer before the next round of sonication. The ratio of seed to uninfected brain homogenate was 1:20 for the first round of PMCA, 1:10 for the second round, and 1:2 for the remaining rounds. Aliquots (n ≥ 3) of uninfected brain homogenate were included in all rounds of PMCA as a negative control.
5
ChIP-PCR Analysis of NOX4 Promoter
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The protein-DNA complexes were crosslinked using 1% formaldehyde, which was then quenched by adding glycine to a final concentration of 200 mM. Chromatin complexes were sonicated to an average size of 250 bp by a MISONIX Sonicator 3000 (Misonix, Farmingdale, NY, USA). The chromatin was incubated with rabbit monoclonal anti‐c-Jun (#61327) (BD Tranduction Laboratory, Los Angeles, CA) and PureProteomeTM Protein A Magnetic Beads (Millipore) overnight. The immunocomplexes were reverse crosslinked, and the purified DNA was subjected to PCR analysis. The PCR primers were listed as follows: NOX4 specific primers (sense, 5'- TGA ATC AGA TGA TGG TCT ACA CTT G -3'; antisense, 5'- AGT GGT CCA AAG GCT TAA CAT TCC -3'). The PCR products were separated by 2% agarose-gel electrophoresis and visualized with ethidium bromide staining.
6
Uniform Isotope Labeling of Recoverin Protein
7
ChIP-qPCR Assay for Histone Modifications
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ChIP assays were performed using previously described methods39 (link). Briefly, ACM from WT and RBPJ cKO mouse hearts were collected, fixed in 2% formaldehyde 12 min at room temperature, rinsed twice in ice-cold PBS, and lysed directly in sonication buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl pH8.0 and protease inhibitors cocktail (Sigma)). The resulting cell suspension was sonicated in a waterbath sonicator (MiSonix Sonicator 3000) using the following programme: 30 s ON, 30 s OFF during10 min (Output Power: 4). The suspension was centrifuged (Eppendorff microfuge) and the cleared supernatant was incubated at 4 °C overnight with an antibody to di-acetylated Histone3 (K9, K18) (Millipore, 07-593) or to tri-methylated Histone3 (K4) (Abcam Ab8580) (both 1 μg/106 cells). Normal rabbit IgG was the negative control (1 μg/106 cells). Protein-DNA complexes were collected on Protein G beads (SIGMA). After rinsing in PBS, the final DNA samples were subjected to PCR. Primer sets specific for the mouse Vegfa, proximal promoters spanning Hif and RBPJ-binding sites were synthesized for this purpose (Supplementary Table 3 ). PCR conditions were 95 °C for 2 min, followed by 25−30 cycles at 95 °C for 30 s, 55−60 °C for 30 s and 72 °C for 30 s.
8
ChIP Assay for Transcription Factors
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The ChIP analysis was performed using the Millipore ChIP kit (Millipore, Billerica, MA, USA), following the manufacturer’s instructions with minor modifications. For each assay, the THP-1 monocytes were inoculated into a 10-cm dish (a total of 5 × 106 cells) and fixed with 1% formaldehyde. Cell pellets were resuspended in SDS lysis buffer containing protease inhibitors (1 mM PMSF, 1 µg/mL aprotinin, and 1 µg/mL pepstatin A). The samples were sonicated with a Misonix sonicator 3000 (Misonix, Farmingdale, NY, USA), centrifuged, and diluted 10-fold in ChIP dilution buffer. After removing an aliquot (whole-cell extract input), the chromatin samples were incubated at 4 °C overnight with antibodies against AP-1 (#9165, Cell Signaling) or NF-κB (#8242, Cell Signaling). The samples were then precipitated by binding to protein A-agarose/salmon sperm DNA beads (Millipore, Billerica, MA, USA). The immunoprecipitated chromatin was analyzed by PCR using primers for the Mac-1 gene promoter. Cycling parameters were 58 °C for 1 min and 95 °C for 30 s, followed by 40 cycles.
9
Multifunctional Antibacterial Nanoparticles
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PLGA-lovastatin-chitosan-tetracycline nanoparticles were prepared by modification of methods reported previously.28 (link) Briefly, 0.12 g PLGA and 4 mg lovastatin were dissolved in chloroform to form a 3 wt% PLGA solution. Chitosan was dissolved in 0.5 M acetic acid and the mixture was then added to the poly(vinyl alcohol) solution containing tetracycline. A solution (W2 phase) containing 0.3 wt% chitosan, 1 wt% poly(vinyl alcohol), and three concentrations (0.1, 0.3, 0.5 wt%) of tetracycline was prepared. Phosphate-buffered saline (PBS) (800 μL) was added to 4 mL of PLGA and the resultant solution was emulsified at 3.5 Hz for 2 minutes using a probe sonicator (Misonix Sonicator 3000, Misonix, Inc., Farmingdale, NY, USA). This W1/O emulsion was then poured into 24 mL of W2 phase and emulsified at 6.5 Hz for 10 minutes. After the evaporation of chloroform, the polymer was precipitated, the nanoparticles were isolated by centrifugation, and then washed with deionized water several times.
10
Coating Flow Cells with Biotinylated DNA
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The flow cells coated with lipid bilayer with attached biotin (DOPC plus DOPE-biotin [1%]) were prepared essentially as described in Han and Mizuuchi, 2010 (link) and rinsed with a buffer containing 25 mM Tris–HCl pH 7.4, 150 mM NaCl, and 5 mM MgCl2 and 0.1 mM CaCl2. Sonicated and biotinylated DNA was prepared as follows: 250 μl of 10 mg/ml sonicated salmon sperm DNA (Sigma) was sonicated for an additional 5 min (Misonix sonicator 3000, output level 6, pulsed on/off for 10 s each at 16°C) to size-weighted average length of ~500 bp. In order to biotinylate the DNA ends, the sonicated DNA (1 mg/ml) was incubated with 40 μM biotin-17-dCTP (Invitrogen) and 0.6 units TdT (NEB) in the buffer specified by the enzyme manufacturer at 37°C for 30 min. The reaction was stopped by heating at 70°C for 10 min, and unincorporated biotin-17-dCTP was removed by using S-200 HR Microspin columns (GE Healthcare). The DNA was ethanol precipitated and resuspended in TE buffer. To coat the flow cell with sonicated DNA, the DNA prepared as above was dissolved to 1 mg/ml in 25 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, and 0.1 mM CaCl2, infused into the assembled flow cell, and incubated overnight at 4°C. Unbound DNA was removed by rinsing with 50 mM Tris–HCl (pH 7.4), 100 mM KCl, 2 mM MgCl2, and 10% glycerol.
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