Nuclei purification without use of detergent was performed following a protocol that we optimized from Blobel & Potter16 (link). Details will be published elsewhere. Typically, human Embryonic Kidney (HEK) 293 T cells were grown on tissue culture dishes in DMEM supplemented with FBS and Streptomycin/Penicillin). After harvesting by trypsinization, cells were incubated in a hypotonic buffer and then lysed by nitrogen cavitation in a cell disruption bomb (Parr Instrument Company, Il. USA). This method prevents alteration of membrane lipid composition as could happen using a classical detergent protocol. Nuclei were purified with a sucrose gradient. Nuclei were further labelled with DiOC6 (1 µM) and Hoechst 33343 (1.6 µM) and observed by confocal fluorescence microscopy to assess the efficiency of nuclei isolation.
Cell disruption bomb
Manufactured by Parr
Sourced in United States
The Cell Disruption Bomb is a laboratory instrument designed to physically disrupt cellular structures. It utilizes high pressure and/or rapid temperature changes to break apart cells, releasing their contents for further analysis or processing.
Automatically generated - may contain errors
6 protocols using cell disruption bomb
1
Detergent-free Nuclei Purification from HEK293T Cells
2
Cell-Free Translation from RACK1-Silenced Extracts
3
Purification and analysis of PHF from AD brain
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Purified, soluble PHF was isolated from AD brain tissue using a protocol modified from the procedure described by Jicha et al. [24 (link)]. Briefly, homogenized AD cortex underwent high pressure batch–gas expansion using a Parr Cell disruption bomb and centrifuged at 28 kg. Soluble PHF was isolated from the supernatant by affinity chromatography using an MC1-Affigel 10 column (25 mL flow rate), with a high guard column (4 cm) of Sepharose 400 Superflow, recycling supernatant twice through the column over 18–20 h at 4 °C. Bound PHF was eluted with two column volumes of 3M potassium thiocyanate. Isolated PHF was analyzed in a tau MC-1 ELISA and a tau AT8 ELISA, which recognizes tau phosphorylated at both Ser 202 and Thr 205 [3 (link)].
4
Toll-Induced Drosomycin Expression in ERTL Cells
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ERTL cells were grown for 5 days at 25°C in 25 ml of culture medium. For the Toll‐induced ERTL cells, the culture medium was supplemented with 2.5 μg/ml recombinant mouse EGF (Sigma‐Aldrich) 16 h before harvesting.
After harvesting, cells were washed two times in cold 40 mM HEPES–KOH pH 8, 100 mM potassium acetate, 1 mM magnesium acetate, and 1 mM DTT solution, and resuspended at a concentration of 109 cells/ml in the same buffer supplemented with 1X Halt™ Protease Inhibitor Cocktail EDTA‐free (Thermo Scientific™). Cell lysis was performed by nitrogen cavitation with a Cell Disruption Bomb (Parr Instrument Company). The lysate was cleared by centrifugations at 4°C with 10,000 g, aliquoted, frozen in liquid nitrogen, and stored at −80°C. The induction of the Toll pathway was checked by monitoring the transcript levels of Drosomycin by RT–qPCR.
After harvesting, cells were washed two times in cold 40 mM HEPES–KOH pH 8, 100 mM potassium acetate, 1 mM magnesium acetate, and 1 mM DTT solution, and resuspended at a concentration of 109 cells/ml in the same buffer supplemented with 1X Halt™ Protease Inhibitor Cocktail EDTA‐free (Thermo Scientific™). Cell lysis was performed by nitrogen cavitation with a Cell Disruption Bomb (Parr Instrument Company). The lysate was cleared by centrifugations at 4°C with 10,000 g, aliquoted, frozen in liquid nitrogen, and stored at −80°C. The induction of the Toll pathway was checked by monitoring the transcript levels of Drosomycin by RT–qPCR.
5
Sucrose Gradient Fractionation of Cell Organelles
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Sucrose gradient was performed as described elsewhere [51 (link)]. Briefly, two 10 cm dishes of A673 and TC71 cells were washed twice with cold PBS and resuspended in 500 μl of homogenization buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L sodium chloride, and 5 mmol/L EDTA), supplemented with 10 μg/mL leupeptin and 10 μg/ml aprotinin. Cells were disrupted at 4°C by nitrogen cavitation in a cell disruption bomb (Parr Instrument Company) at 800 psi for 15 minutes and collected dropwise. Afterward, cells were passed back and forth through a 22-gauge needle 25 times at 4°C. Nuclei and unbroken cells were removed by centrifugation at 1,600 g in a MLA-130 rotor (Beckman Coulter) for 5 minutes at 4°C. The resulting supernatant (500 μL) was mixed with an equal volume of 2.5 M sucrose and loaded at the bottom of a discontinuous sucrose gradient formed by layers of 200 μL of 30, 25, 20, 15, 10, and 5% sucrose (wt/vol) freshly prepared in homogenization buffer. Gradients were centrifuged in a TLS-55 swinging-rotor (Beckman Coulter) without brake at 166,000 g for 3 h at 4°C. Five fractions of 280 μl were collected from the top in addition to a final lower fraction of 700 μL by using a CentriTube Slicer (Beckman Coulter). The gradients shown in the figures are representative of at least two independent experiments.
6
Drosophila S2 Cell In Vitro Translation Assay
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In vitro translation competent extracts from Drosophila melanogaster S2 cells were prepared as previously described (25 (link),26 (link)). Cells were lysed in 40 mM HEPES–KOH [pH8], 100 mM potassium acetate, 1 mM magnesium acetate, 1 mM DTT at a density of 109 ml−1 using a Cell Disruption Bomb (Parr Instrument Company). The lysate was then cleared by centrifugations at 4°C and supplemented with creatine kinase at 0, 24°C, aliquoted and stored at −80°C. In vitro translation experiments were performed as previously described under subsaturating conditions to avoid substrates titration (25 (link),26 (link)). Translation efficiency was determined by Renilla Luciferase assay. RNA integrity of translated reporter mRNAs was checked by polyacrylamide gel electrophoresis (PAGE) on denaturing 4% acrylamide gels.
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In vitro translation competent extracts from Drosophila melanogaster S2 cells were prepared as previously described (25 (link),26 (link)). Cells were lysed in 40 mM HEPES–KOH [pH8], 100 mM potassium acetate, 1 mM magnesium acetate, 1 mM DTT at a density of 109 ml−1 using a Cell Disruption Bomb (Parr Instrument Company). The lysate was then cleared by centrifugations at 4°C and supplemented with creatine kinase at 0, 24°C, aliquoted and stored at −80°C. In vitro translation experiments were performed as previously described under subsaturating conditions to avoid substrates titration (25 (link),26 (link)). Translation efficiency was determined by Renilla Luciferase assay. RNA integrity of translated reporter mRNAs was checked by polyacrylamide gel electrophoresis (PAGE) on denaturing 4% acrylamide gels.
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