Fibroblast mitochondria were isolated by differential centrifugation [22] (link). Before trypsinization, the cultured cells were washed with chilled PBS at least four times to make sure that there was no residual FBS. The cultured fibroblasts were then trypsinized with 0.25% trypsin-EDTA. Samples of 0.5×106 cells were suspended in 1 ml of isolation buffer containing 0.25 M sucrose, 10 mM HEPES (pH 7.5) and 0.1 mM EDTA. Cell suspensions were sonicated with a probe sonicator (Bandelin Sonopuls HD 200; Bandelin electronic; Berlin, Germany) at MS 72/D (50 cycles) for 10 sec. Cell lysates were then centrifuged twice at 1,000 g for 10 min to remove cell debris and intact cells, if present. Supernatant was collected and centrifuged at 20,000 g for 30 min. Pellets were saved and washed with the buffer containing 0.25 M sucrose and 10 mM HEPES (pH 7.5) and centrifuged again at 20,000 g for 20 min. As a final step, the mitochondrial pellets were washed with PBS and centrifuged at 20,000 g for 10 min. The whole extraction procedure was performed at 4°C. The mitochondrial pellets were lyzed by Laemmli or 2-DE buffer depending on the subsequent step of the experiment, and the lyzed mitochondrial fractions were kept at −20°C until use.
Sonopuls hd 200
Manufactured by Bandelin
Sourced in Germany
The Sonopuls HD 200 is a compact ultrasonic device designed for laboratory applications. It generates high-frequency sound waves to aid in various processes, such as cell disruption, homogenization, and emulsification. The device features an adjustable power output and a timer to control the duration of the ultrasonic treatment.
Automatically generated - may contain errors
Lab products found in correlation
Hw 200, by Bandelin (2 mentions)
Complete protease inhibitor mix, by Roche (1 mentions)
Pierce streptavidin magnetic beads, by Thermo Fisher Scientific (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
M082301, by Agilent Technologies (1 mentions)
Eclipse 50i epifluorescence microscope, by Nikon (1 mentions)
Ethanol, by Carl Roth (1 mentions)
13 protocols using sonopuls hd 200
1
Fibroblast Mitochondria Isolation Protocol
2
Ultrasonic Bath and Sonicator Optimization for Biological Samples
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An electronic ultrasonic bath (8510-DTH, BRANSON CO. LTD. Brookfield, USA) was used to sonicate the JAE. JAE (10 mL) in falcon tubes was immersed in an electronic ultrasonic bath and treated for 10, 30, 60, and 180 s.
An electronic sonicator (Bandelin sonopuls HD 200, Bandelin Electronic, Berlin, Germany) was used to directly sonicate on JAE. The amplitude and time conditions were set at 30, 60, and 90% with 30, 60, and 120 s. After sonication, the JAEs were sterilized and filtered using the same method as described previously.
An electronic sonicator (Bandelin sonopuls HD 200, Bandelin Electronic, Berlin, Germany) was used to directly sonicate on JAE. The amplitude and time conditions were set at 30, 60, and 90% with 30, 60, and 120 s. After sonication, the JAEs were sterilized and filtered using the same method as described previously.
3
Proximity-Based Protein Identification
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U2OS cell lines, stably transfected with inducible BirA*-LEM2, BirA*-MAN1, BirA*-emerin and BirA*-GFP, were incubated as described above. After three washing steps with 1 × DPBS, cells were scraped off and lysed on ice in (1 ml / 15 mm petri dish) high-salt RIPA buffer (25 mM Tris-HCl pH 7.4, 500 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS), supplemented with 1 x complete protease inhibitor mix (Roche, Vienna, Austria). After sonication (Bandelin Sonopuls HD200; power MS 73/D, continuous pulse), 100 µl Pierce streptavidin magnetic beads (Thermo Scientific, Vienna, Austria) were added to the lysate and incubated overnight at 4 °C. Magnetic beads were collected and washed extensively with the following washing buffers in sequence—twice with high-salt RIPA buffer, twice with wash buffer A (2% SDS in ddH2O), once with wash buffer B (0.1% deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM Hepes, pH 7.5), once with wash buffer C (250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 10 mM Tris, pH 8.1), twice with wash buffer D (50 mM Tris, pH 7.4, and 50 mM NaCl) at 4 °C. 10% of the samples were used for Western blot analysis and 90% were analyzed by mass spectrometry after on-bead tryptic digest.
4
FISH-based Microbial Community Analysis
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Sediment samples from the untreated core, untreated-3 years core and SOFT core have been fixed in 3% formaldehyde for 3 h at 4°C, washed with 1x PBS and stored in ethanol-PBS (1:1) at -20°C. Samples were diluted, four times ultrasonicated on ice at 20% intensity, 20 cycles, 30 s (Bandelin Sonopuls HD200), and filtered on a 0.22 μm pore size polycarbonate filter. In situ hybridizations with horseradish peroxidase (HRP)-labeled probes followed by fluorescently labeled tyramide signal amplification (catalyzed reporter deposition) were carried out as described previously (Pernthaler et al., 2002 (link)). Permeabilization was performed by lysozyme treatment (10 mg ml-1) for 60 min at 37°C. Hybridization was done at 46 °C. Hybridized samples were analyzed with an epifluorescence microscope (Nikon Eclipse 50i). For each probe and sample, >1000 DAPI stained cells and their corresponding FISH signals were counted. Used probes (ordered from biomers.net; Ulm, Germany) and formamide concentrations are given in Supplementary Table S1 .
5
Metabolomic Analysis of Aortic SMCs
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Primary SMCs were isolated from aneurysmal and control tissue of the ascending aorta as described in more detail elsewhere (11 (link), 19 (link)). Cells were cultivated in specific SMC medium (Medium 231 supplemented with SMC Growth Supplement; Gibco, Thermo Fisher Scientific, Austria) at 37°C until reaching passage 3 before being used for experiments. All SMC samples were tested for contamination with endothelial cells and fibroblasts by Western blot analyses using an anti-CD90 antibody for fibroblasts (Dianova, Germany, #DIA-100) and an anti-CD31 antibody for endothelial cells (Dako, United States, #M082301).
For metabolomic analyses, trypsinized cells were counted and 1x106 cells per sample were added to 100 μl ice cold ultrapure absolute Ethanol (Carl Roth, Karlsruhe, Germany) for cell lysis. Samples were sonicated (15 intervals, cycles: 10%, power: MS72/; Sonopuls HD 200; Bandelin, Berlin, Germany) and all solid components were separated by centrifugation at 10,800 g at 4°C for 10 min. Supernatants were collected and stored at −80°C for further analyses.
For metabolomic analyses, trypsinized cells were counted and 1x106 cells per sample were added to 100 μl ice cold ultrapure absolute Ethanol (Carl Roth, Karlsruhe, Germany) for cell lysis. Samples were sonicated (15 intervals, cycles: 10%, power: MS72/; Sonopuls HD 200; Bandelin, Berlin, Germany) and all solid components were separated by centrifugation at 10,800 g at 4°C for 10 min. Supernatants were collected and stored at −80°C for further analyses.
6
Optimizing Cell Lysis through Freezing, Sonication
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To enhance cell lysis samples were subjected to different disintegration strategies: (i) 3 cycles of freezing (1 h at −80 °C) and thawing (30 min at 37 °C) after the bead-beating step, (ii) sonic treatment using a 100 W ultrasonic bath (Elmasonic, Elma), and (iii) sonic treatment (200 W) with a special sonication device (Bandelin Sonopuls HD 200 equipped with a HW 200). The sonication was repeated three times for 60 s, 40 s, and 20 s, respectively, in the ultrasonic bath (100 W) and for 30 s each for the sonication device (200 W).
7
Epifluorescence Microscopy of Sediment Microbes
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Sediment samples were fixed in 3% formaldehyde for 3 h at 4°C, washed with 1x phosphate-buffered saline (PBS) and stored in ethanol-PBS (1:1) at -20°C. Samples were diluted, four times ultrasonicated on ice at 20% intensity, 20 cycles, 30 s (Bandelin Sonopuls HD200). An aliquot was filtered on a 0.22 μm pore size polycarbonate filter. Filter sections were embedded in Citifluor:Vectashield (4:1) mounting medium containing 50 μg ml-1 DAPI. Microscopy was done with a Nikon eclipse 50i epifluorescence microscope.
8
Metabolite Extraction from Aortic Tissue
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Following surgical extraction, cleaning, and rinsing of ascending thoracic aortic tissue samples, samples were immediately snap frozen in liquid nitrogen and stored at -80°C until further use. For metabolite extraction, per sample 40 mg of aortic tissue were placed in sterile polystyrene tube on dry ice, followed by adding 6 μl ice cold 100% ethanol (ultrapure; Carl Roth, Karlsruhe, Germany) per mg of tissue. Consequently, a 5 mm diameter hardened steel ball (Retsch, Haan, Germany) was added into the tubes, and tubes were placed in a Mixer Mill MM400 (Retsch, Haan, Germany), followed by breaking up of tissue pieces by applying a vibrational frequency of 30 Hz for 3 minutes. In the next step samples were sonicated (15 intervals, cycles: 10%, power: MS72/D; Sonopuls HD 200, Bandelin, Berlin, Germany) and by a centrifugation step (10 min/20,800 x g/4°C) all solid components were separated. Supernatants were collected and stored on dry ice until metabolite analyses.
9
Enumeration of Bacterial Cell Cultures
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Cells of the D. shibae cultures were enumerated by flow cytometry, those of the P. inhibens cultures by epifluorescence microscopy due to the formation of microaggregates. Flow cytometric analyses were done according to Osterholz et al. (2015 (link)). Cell aggregates of P. inhibens were dispersed by ultrasonication (5 × 10 s at 15 mV; Bandelin Sonopuls HD 200, Bandelin, Berlin, Germany), filtered onto a 0.2 μm polycarbonate membrane, stained for 30 min with SYBR® Green I and counted by epifluorescence microscopy as described (Lunau et al., 2005 (link)). At least 1,000 cells were enumerated per filter.
10
Cell Lysis Optimization through Disintegration Strategies
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