Fresh-frozen human post-mortem CTE (Brodmann area 8/9; superior frontal cortex) and HC (frontal cortex) brain tissues were homogenized to a concentration of 100 mg/ml (wet weight). Tissues were mechanically homogenized on ice in ice-cold 50 mM Tris–HCl buffered saline (TBS) (pH 7.4) using a dounce homogenizer (Fisher Scientific). Homogenates were snap frozen and stored at −80°C.
Dounce homogenizer
Manufactured by Thermo Fisher Scientific
Sourced in Canada, United States
The Dounce homogenizer is a manual laboratory device used for gently disrupting cells and tissues to extract their contents, such as proteins, organelles, or nucleic acids. It consists of a glass or Teflon pestle that fits snugly inside a glass mortar, allowing for controlled and gentle homogenization of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Tla100 rotor, by Beckman Coulter (2 mentions)
Formaldehyde, by Thermo Fisher Scientific (1 mentions)
Glycine, by Merck Group (1 mentions)
Dpbs buffer, by Lonza (1 mentions)
Hindiii, by New England Biolabs (1 mentions)
Neb buffer 2, by New England Biolabs (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Tla 55 rotor, by Beckman Coulter (1 mentions)
Np40 cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Specific quantitative sandwich elisa kits, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Genomic lysis buffer, by Zymo Research (1 mentions)
Zymo spin 3 column, by Zymo Research (1 mentions)
E1015, by Applygen (1 mentions)
P1511, by Applygen (1 mentions)
11 protocols using dounce homogenizer
1
Homogenization of Post-Mortem Brain Tissues
2
Mitochondria Isolation from Bronchial Cells
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Isolation of mitochondria from cell pellets was achieved using a differential centrifugation procedure [35 (link)]. The pellet of bronchial cells was resuspended in 250 µL of mitochondrial isolation buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl, 1 mM EDTA, pH 7.5) and homogenized with 15 strokes in a Dounce homogenizer (Fisher Scientific, France) on ice. To obtain mitochondria, the homogenate was subjected to differential centrifugation steps as described previously [35 (link)].
Mitochondrial purity and efficacy of isolation were assessed by the determination of lactate dehydrogenase (LDH) and glutamate dehydrogenase (GDH) activities.
Mitochondrial purity and efficacy of isolation were assessed by the determination of lactate dehydrogenase (LDH) and glutamate dehydrogenase (GDH) activities.
3
Extraction of Water-Soluble Amyloid-Beta
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Homogenates of human brains were prepared as described elsewhere [17 (link)]. Frozen cortices were provided by Dr M Frosch (MGH and MADRC Neuropathology Core, Harvard, MA, USA) under institutional review board-approved protocols. Frozen samples of temporal or frontal cortex (1 g) were allowed to thaw on ice, chopped into small pieces with a razor blade, and then homogenized with 25 strokes of a Dounce homogenizer (Fisher, Ottawa, ON, Canada) in 4 ml ice-cold 20 mM Tris–HCl, pH 7.4, containing 150 mM NaCl (Tris-buffered saline (TBS)) and protease inhibitors. Water-soluble Aβ was separated from membrane-bound and plaque Aβ by centrifugation at 175,000 × g and 4°C in a TLA 100 rotor (Beckman Coulter, Fullerton, CA, USA) for 30 minutes, and the supernatant (referred to as TBS extract) aliquoted and stored at −80°C. The ethical body approving this study was the Partners Institutional Review Board of the Partners Human Research Committee.
4
Chromatin Crosslinking and Digestion for Hi-C
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In brief, 20–25 million Se-Ax cells (24 (link)) were cross-linked with formaldehyde (Thermo Fisher Scientific, Waltham, MA, USA). Crosslinking was stopped by addition of 125 mM glycine (Merck Millipore, Darmstadt, Germany). After washing cells in ice-cold DPBS buffer (Lonza, Basel, Switzerland), cell pellets were flash frozen and stored at −80°C. For lysis, cells were resuspended in Hi-C lysis buffer and lysed using a Dounce homogenizer (Fisher Scientific GmbH, Schwerte, Germany). After centrifugation, pellets were washed twice in NEB buffer 2 (New England Biolabs, Ipswhich, MA, USA), finally resuspended in 370 µl NEB buffer 2 and 50 µl were transferred to seven tubes each. In order to remove proteins not cross-linked to DNA, 38 µl 1%SDS (Sigma-Aldrich, St. Louis, MO, USA) was added to each tube and incubated for 10 min at 65°C. Afterward, SDS was inactivated by the addition of 44 µl 10% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). In each but one tube 400 U HindIII (New England Biolabs, Ipswhich, MA, USA) were added and DNA was digested overnight at 37°C with rotation. The next day, the tube with undigested DNA and one HindIII treated sample were removed to verify HindIII digestion efficiency.
5
Extraction of Water-soluble Aβ from Human Brains
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Homogenates of human brains were prepared as described elsewhere [64 (link), 81 (link)]. Frozen brain tissue collected at Massachusetts Alzheimer’s Disease Research Center (MADRC Neuropathology Core, Harvard, MA, USA) and Brigham and Women’s Hospital under institutional review board-approved protocols. Frozen samples of temporal or frontal cortex (1 g) were allowed to thaw on ice, chopped into small pieces with a razor blade, and then homogenized with 25 strokes of a Dounce homogenizer (Fisher, Ottawa, ON, Canada) in 4 ml ice-cold 20 mM Tris–HCl, pH 7.4, containing 150 mM NaCl (Tris-buffered saline (TBS)) and protease inhibitors. Water-soluble Aβ was separated from membrane-bound and plaque Aβ by centrifugation at 175,000×g and 4 °C in a TLA 100 rotor (Beckman Coulter, Fullerton, CA, USA) for 30 min, and the supernatant (referred to as TBS extract) aliquoted and stored at − 80 °C. The ethical body approving this study was the Partners Institutional Review Board of the Partners Human Research Committee.
6
Aβ Extraction and Immunodepletion from Human Brain
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The same human brain tissue was used as our previous report (17) . Human brain tissue was used in accordance with local Ethics Committee guidelines. Brie y, tris-buffered saline (TBS) extracts of brain specimens were prepared, processed and analyzed as described previously. Frozen cortex (0.9 g) was allowed to thaw on ice, chopped into small pieces and homogenized in 4.5 ml of ice-cold 20 mM Tris-HCl, pH 7.4, containing 150 mM NaCl with 25 strokes of a Dounce homogenizer (Fisher, Ottawa, Ontario, Canada). Water-soluble Aβ was separated from membrane-bound and plaque Aβ by centrifugation at 91,000g and 4 o C in a TLA 55 rotor (Beckman Coulter, Fullerton, CA, USA) for 78 minutes. To eliminate bioactive small molecules the supernatant was exchanged into ammonium acetate. Thereafter, extracts were divided into 2 parts: one aliquot was immunodepleted of Aβ by 3 rounds of 12-h incubations with the anti-Aβ antibody, AW8, and protein A at 4 o C. The second portion was not manipulated in any way and is simply referred to as AD. Aliquots of samples were stored at -80 o C .
7
Cytokine Quantification from Brain Tissue
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Ipsilateral brain was homogenized using Dounce Homogenizer in 10 volumes of NP40 cell lysis buffer (FNN0021; Thermo Fisher Scientific) supplemented with 1 mm phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail (Sigma‐Aldrich). All steps were carried out at 4 °C. The homogenate was centrifuged initially at 700 g for 5 min to eliminate unruptured cells and debris and then further centrifuged at 12 500 g for 20 min, and the supernatant was used to measure cytokine levels by ELISA. Tumor necrosis factor‐alpha (TNF‐α), IL‐1β, interleukin‐10 (IL‐10) and IL‐4 levels were measured by commercially available specific quantitative sandwich ELISA kits according to the manufacturer's instructions (eBioscience). The cytokine levels were normalized by total protein.
8
Genomic DNA Extraction from Fall Armyworm
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Specimens were obtained as larvae from corn (maize) or sorghum plants at various locations in northern Sub-Saharan Africa at various times in 2016 (Table 1 ). Specimens were stored either air-dried or in ethanol at room temperature. A portion of each specimen was excised and homogenized in a 5-ml Dounce homogenizer (Thermo Fisher Scientific, Waltham, MA, USA) in 800 µl Genomic Lysis buffer (Zymo Research, Orange, CA, USA) and incubated at 55 °C for 5–30 min. Debris was removed by centrifugation at 10,000 rpm for 5 min. The supernatant was transferred to a Zymo-Spin III column (Zymo Research, Orange, CA, USA) and processed according to manufacturer’s instructions. The DNA preparation was increased to a final volume of 100 µl with distilled water. Genomic DNA preparations of fall armyworm samples from previous studies were stored at −20 °C.
9
Mitochondrial Fractionation from Adipocytes
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Mitochondrial and cytosolic fractions were isolated freshly from differentiated sWACs (∼10 × 106 cells). All steps were performed on ice or at 4°C. Cells were trypsinized, centrifuged at 1200 rpm, and pellets were resuspended in 1 ml HES buffer [20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 1 mM EDTA, 250 mM sucrose in ddH2O, pH 7.4] containing 1× PIC mixture and 1× phosphatase inhibitor (PI) mixture (0.1 M Na-orthovanadate, 50 mM Na-glycerophosphate, 0.5 M Na-fluoride). Cell homogenization was performed using a Dounce homogenizer (60 strokes; Thermo Fisher Scientific). Cell debris (500 g, 5 min) and nuclei (1000 g, 10 min) were removed by centrifugation. The resulting supernatant was centrifuged at 13,000 g for 20 min to yield a crude mitochondrial pellet. The pellet was washed 3 times in 1 ml HES buffer containing 1× PIC and 1× PI and was centrifuged again at 13,000 g for 20 min. Cell debris (cp), nuclear (nuc), and mitochondrial fraction were resuspended in RIPA buffer + 1× PIC + PI. Cytosolic proteins were precipitated in 100% ice-cold acetone (4/1, v/v) for 2 h at −20°C. Samples were centrifuged at 15,000 g for 20 min. Resulting pellet was dried for 10 min at room temperature and resuspended in 150 µl RIPA buffer + 1× PIC + PI.
10
Potato Mitochondria Isolation Protocol
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Mitochondria were isolated from potato tubers in a medium containing 225 mM of sucrose, 100 mM of mannitol, 20 mM of HEPES, 1 mM of EDTA, 1 mM of DTT, and1.5 mg/L of fatty acids free BSA, pH 7.4 (KOH). The tubers of potatoes were homogenized with Dounce homogenizer (Thermo Fisher Scientific, USA) in the medium in a 1:1 ratio at +4 °C. The homogenate was centrifuged for 5 min at 1000× g. The resulting supernatant was transferred into a clean centrifuge tube and centrifuged for 5 min at 4000× g. The resulting supernatant was separated from the pellet, transferred to the new centrifuge tube, and centrifuged for 15 min at 15,000× g. Then, mitochondria were purified by centrifugation in a 17% Percoll gradient for 30 min at 30,000× g with a centrifuge acceleration of 6. After centrifugation, the mitochondrial fraction located 1 cm from the bottom (3–4 mL) was collected, diluted with the medium, and centrifuged for 15 min at 18,000× g. The pellet was transferred to a 1.5 mL Eppendorf tube and centrifuged for 10 min at 16,000× g. The supernatant was removed and the pellet was resuspended in 150 μL of isolation medium.
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