αSyn transduction into mouse primary hippocampal neurons was performed as described previously32 (link). hPFF, LB-αSyn, ampLB, and αSyn aggregates extracted from Thy1:SNCA/Snca–/– mouse brains were diluted in DPBS and sonicated with a Diagenode Biorupter sonicator (20 min, 30 s on, 30 s off, 10 °C, high setting). Neurons were then treated with a noted dose of the αSyn preparations at 7 days in vitro (DIV), fixed, and immunostained at 14 days post-treatment (21 DIV). The amounts of αSyn transduced in Fig. 1f , Supplementary Fig. 1, and in Supplementary Fig. 4e were 25 and 50 ng/well, respectively. For the treatment with αSyn aggregates extracted from Thy1:SNCA/Snca–/– mouse brains in Fig. 7b , the brain lysates containing 25 ng of αSyn or up to 6.4 μg of total protein per well were transduced to avoid significant toxicity of contaminants for cultured neurons32 (link). The resulting amounts of αSyn were 2.4 ng/well (Thy1:SNCA/Snca–/– mice without αSyn pathology), 19 ng/well (ampLB-injected Thy1:SNCA/Snca–/– mice), and 25 ng/well (hPFF-injected Thy1:SNCA/Snca–/– mice and Thy1:SNCA/Snca–/– mice with spontaneous αSyn pathology).
Biorupter sonicator
Manufactured by Diagenode
The Biorupter sonicator is a lab equipment product from Diagenode. It is a device used to break apart biological samples, such as cells or tissues, through the application of high-frequency sound waves. The Biorupter sonicator is designed to disrupt the samples for various downstream applications, such as protein or DNA extraction.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Nupage novex 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Nupage sds page gel system, by Thermo Fisher Scientific (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Hrp conjugated secondary ab, by Bio-Rad (1 mentions)
G box chemi imaging system, by Syngene (1 mentions)
Model 680 microplate reader, by Bio-Rad (1 mentions)
Nr0b1 antibody, by Cell Signaling Technology (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
10 protocols using biorupter sonicator
1
Transduction of α-Synuclein into Hippocampal Neurons
2
Cpf1 Protein Expression and Purification
3
HEK293T Chromatin Immunoprecipitation Assay
4
Western Blot Analysis of Naive B Cells
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All antibodies used for western blotting are listed in Supplementary Table 3 . 0.8–1 × 106 primary naive B cells were recovered for protein extraction, which was performed using RIPA lysis buffer (Pierce) followed by cycles of sonication performed using the Biorupter Sonicator (Diagenode). Protein concentration was analysed using the BCA Protein Assay kit (cat no. 23225 from Pierce) and read by the Model 680 Microplate reader (Bio-Rad). Proteins were separated by the NuPAGE SDS-PAGE Gel system (Thermo Scientific) and transferred onto Immobilon-P PVDF membranes (Sigma-Aldrich) according to standard procedure. Detections were performed with HRP-conjugated secondary Ab (Bio-Rad) and enhanced chemiluminescent (ECL Plus) reagent (Amersham) using the G:BOX Chemi imaging system (Syngene). GAPDH or β-ACTIN on the same membrane served as loading control. Uncropped original scans of immunoblots are provided in Supplementary Fig. 9 .
5
Partial Proteinase K Digestion of α-Synuclein
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Sarkosyl-insoluble fractions from LBD brains and Thy1:SNCA/Snca−/− mouse brains were prepared by biochemical brain extraction. For partial PK digestion, 50 ng of αSyn from each sample was sonicated with a Diagenode Biorupter sonicator (20 min, 30 s on, 30 s off, 10 °C, high setting) and mixed with 0.2 μg of PK in DPBS to a final volume of 50 μl and incubated at 37 °C for 1, 5, 15, and 30 min. The reaction was stopped with 1 mM PMSF. The samples were boiled with SDS-sample buffer for 10 min and resolved on NuPAGE Novex 12% Bis-Tris gels (Invitrogen). Transferred nitrocellulose membranes were probed with an anti-human αSyn antibody HuA (CNDR, 1:500) and an anti-αSyn antibody Syn1 (BD transduction #610787, 1:500).
6
ChIP-seq Profiling of NR0B1 Transcription Factor
7
Partial Proteinase K Digestion of α-Synuclein
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Sarkosyl-insoluble fractions from LBD brains and Thy1:SNCA/Snca–/– mouse brains were prepared by biochemical brain extraction. For partial PK digestion, 50 ng of αSyn from each sample was sonicated with a Diagenode Biorupter sonicator (20 min, 30 s on, 30 s off, 10 °C, high setting) and mixed with 0.2 μg of PK in DPBS to a final volume of 50 μl and incubated at 37 °C for 1, 5, 15, and 30 min. The reaction was stopped with 1 mM PMSF. The samples were boiled with SDS-sample buffer for 10 min and resolved on NuPAGE Novex 12% Bis–Tris gels (Invitrogen). Transferred nitrocellulose membranes were probed with an anti-human αSyn antibody HuA (CNDR, 1:500) and an anti-αSyn antibody Syn1 (BD transduction #610787, 1:500).
8
Transduction of α-Synuclein in Hippocampal Neurons
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αSyn transduction into mouse primary hippocampal neurons was performed as described previously32 (link). hPFF, LB-αSyn, ampLB, and αSyn aggregates extracted from Thy1:SNCA/Snca−/− mouse brains were diluted in DPBS and sonicated with a Diagenode Biorupter sonicator (20 min, 30 s on, 30 s off, 10 °C, high setting). Neurons were then treated with noted dose of the αSyn preparations at 7 d in vitro (DIV), fixed and immunostained at 14 d post-treatment (21 DIV). The amount of αSyn transduced in Figures 1F , S1, and S6C was 25 ng/well. For the treatment with αSyn aggregates extracted from Thy1:SNCA/Snca−/− mouse brains in Figure 7B , the brain lysates containing 25 ng of αSyn or up to 6.4 μg of total protein per well were transduced to avoid significant toxicity of contaminants for cultured neurons32 (link). The resulting amounts of αSyn were 2.4 ng/well (Thy1:SNCA/Snca−/− mice without αSyn pathology), 19 ng/well (ampLB-injected Thy1:SNCA/Snca−/− mice), and 25 ng/well (hPFF-injected Thy1:SNCA/Snca−/− mice and Thy1:SNCA/Snca−/− mice with spontaneous αSyn pathology).
9
Membrane-bound Catalase Activity Assay
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The catalase activity in membranes was determined by following the oxygen production (see above) in standard buffer without detergent at different H2O2 concentration. To test whether the catalase activity was membrane-associated, the membranes of E. coli overexpressing cytochrome bd were washed by first diluting the membrane suspension (1:13) in standard buffer without detergent. The diluted suspension was sonicated (10 min in a Biorupter sonicator from Diagenode at maximum intensity) to disrupt possible membrane vesicles containing cytosolic proteins. The sonicated membrane suspension was centrifuged for 1 h at 300,000 g for membrane recovery. The membrane pellet was resuspended in buffer prior to the polarographic assay. The dilution/sonication procedure was repeated (second wash) using the product from the first step and the polarographic assay was performed again.
10
HEK293T Chromatin Immunoprecipitation Assay
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HEK293T cells were transfected with 40 μg total plasmid in 15cm
dishes. After three days, cells were fixed in 1% formaldehyde for 10 min at room
temperature. The reaction was quenched with 0.125M glycine and the cells were
lysed using Farnham lysis buffer with a protease inhibitor cocktail (Roche).
Nuclei were collected by centrifugation at 2,000rpm for 5min at 4°C and
lysed in RIPA buffer with a protease inhibitor cocktail (Roche). Chromatin was
sonicated using a Biorupter Sonicator (Diagenode, model XL) and
immunoprecipitated using anti-Flag (Sigma, M2). The formaldehyde crosslinks were
reversed by heating overnight at 65°C and genomic DNA fragments were
purified using a spin column. For qPCR, 500 pg of ChIP’d DNA was used per
reaction. qPCR was performed as described above. The data are presented as fold
change gDNA normalized to a region of the β-actin locus and relative to
samples targeting Cascade with the control crRNA mentioned above. All sequences
for qPCR primers can be found inSupplementary Table S4 .
dishes. After three days, cells were fixed in 1% formaldehyde for 10 min at room
temperature. The reaction was quenched with 0.125M glycine and the cells were
lysed using Farnham lysis buffer with a protease inhibitor cocktail (Roche).
Nuclei were collected by centrifugation at 2,000rpm for 5min at 4°C and
lysed in RIPA buffer with a protease inhibitor cocktail (Roche). Chromatin was
sonicated using a Biorupter Sonicator (Diagenode, model XL) and
immunoprecipitated using anti-Flag (Sigma, M2). The formaldehyde crosslinks were
reversed by heating overnight at 65°C and genomic DNA fragments were
purified using a spin column. For qPCR, 500 pg of ChIP’d DNA was used per
reaction. qPCR was performed as described above. The data are presented as fold
change gDNA normalized to a region of the β-actin locus and relative to
samples targeting Cascade with the control crRNA mentioned above. All sequences
for qPCR primers can be found in
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