After decapitation, the mouse brain was removed and homogenized using a KIMBLE Dounce tissue grinder (Sigma-Aldrich, USA) in buffer A, containing 225 mM mannitol (Sigma-Aldrich), 75 mM sucrose (Dia-M, Russia), 5 mM Hepes (BioClot, Germany), 1 mM ethylene glycol tetraacetic acid (EGTA) (Sigma-Aldrich), pH 7.4 with the addition of 2 mg/mL fatty acid-free bovine serum albumin (BSA) (Dia-M). The wash buffer (buffer B) used in the centrifugation step had the same composition except BSA. The resulting homogenate was centrifuged using a Z 36 HK centrifuge (Hermle Labortechnik, Germany) for 5 min at 900 g. The supernatant was transferred into clean tubes and centrifuged for 10 min at 14 000 g. After that, the supernatant was removed, and the pellet was resuspended in 100 µL of buffer B and after addition of digitonin (Sigma-Aldrich) (0.02% final concentration) the susnepsion was incubated on ice for 2 min. The tubes were centrifuged for 15 min at 14 000 g. The supernatant was removed again, the pellet was resuspended in 100 µL of buffer B, and then centrifuged for 10 min at 14 000 g. The last step was repeated twice. The resulting pellet was resuspended in 20 µL of buffer B [22 (link)].
Kimble dounce tissue grinder
Manufactured by Merck Group
Sourced in United States
The KIMBLE Dounce tissue grinder is a laboratory equipment used for homogenizing and disrupting cells, tissues, and other biological samples. It features a glass pestle that fits snugly into a glass cylinder, allowing for gentle yet effective mechanical disruption of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Ethylene glycol tetraacetic acid (egta), by Merck Group (2 mentions)
Mannitol, by Merck Group (2 mentions)
Sucrose, by Dia-M (2 mentions)
Z 36 hk centrifuge, by Hermle (2 mentions)
Nuclei ez lysis buffer, by Merck Group (2 mentions)
Digitonin, by Merck Group (1 mentions)
Qiaquick nucleotide removal kit, by Qiagen (1 mentions)
Qproteome mitochondria isolation kit, by Qiagen (1 mentions)
Cell strainer snap cap, by BD (1 mentions)
Mitochondria isolation kit for tissue, by Abcam (1 mentions)
Halt protease inhibitor single use cocktail 100x, by Thermo Fisher Scientific (1 mentions)
7 protocols using kimble dounce tissue grinder
1
Isolation of Mouse Brain Mitochondria
2
Mitochondrial DNA Isolation and Analysis
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Mitochondrial DNA was isolated from cytosolic fraction using the Qproteome mitochondria isolation kit (Qiagen, Cat No # 37612) with minor modifications. Briefly, 5 × 106 BMDMs from Irgm1+/+ and Irgm1−/− mice were seeded in 10 cm dish. Next day, cells were harvested and washed twice with ice‐cold 1× PBS and once with 0.9% sodium chloride solution at 500 g for 5 min at 4°C. The cell pellet was resuspended in 500 μl lysis buffer and homogenized using KIMBLE Dounce tissue grinder (Sigma, Cat. No # D8938) with 8–10 strokes on ice. The cell suspension was centrifuged at 700 g for 10 min at 4°C and again at 7,000 g for 10 min at 4°C, and the supernatant was collected. The total DNA was isolated from the supernatant (cytosolic fraction) by using the QIAquick nucleotide removal kit (Qiagen, Cat No. 28304) and eluted in 25 μl elution buffer. qRT–PCR was performed using cytochrome c oxidase subunit 1 primer. For normalization of the assay, 18S RNA primer was used.
3
Nuclei Extraction and Preservation Protocol
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Fixed root tips or anthers cut with a razor blade were transferred into a 2 mL KIMBLE Dounce tissue grinder equipped with pestles A (large clearance) and B (small clearance) (Sigma-Aldrich, D8938; [44 (link)]) followed by adding 1 mL of LB01 buffer (15 mM Tris–HCl, 20 mM NaCl, 80 mM KCl, 0.5 mM spermine, 2 mM Na2EDTA, and 0.1% (v/v) Triton X-100, pH 9.0) [20 (link)]. The tissues were homogenised with pestle A for one min and for an additional four minutes with pestle B, which resulted in a homogeneous cell suspension, lacking an apparent debris. The suspension was then filtered through a 70-µm and 40-µm cell strainer (pluriStrainer Mini 70 µm, 43-10070-40 and pluriStrainer Mini 40 µm, 43-10040-40; pluriSelect Life Science, Leipzig, Germany). The filtered cell suspension was centrifuged at 2000 × g and 4 °C for 5 min. The supernatant was removed, and the pellet was resuspended in 50 µL of LB01 buffer. Five to eight µL of cell suspension were pipetted onto adhesion microscope slides (Epredia Superfrost® Plus Adhesion Microscope Slides; Menzel-Gläser, Braunschweig, Germany) and allowed to air dry (8–10 min). The specimens were either used directly for the cytological experiments or frozen on dry ice and stored for several weeks or months at – 80 °C.
4
Nuclear Isolation and FACS Profiling
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Neuronal nuclei were isolated from dissected brain as described86 (link). Briefly, tissue from the MC and HPC were dissected and homogenized in 2 mL of Nuclei EZ Lysis Buffer (Sigma-Aldrich) using a KIMBLE Dounce Tissue Grinder (Sigma-Aldrich). After the addition of an additional 2 mL of Nuclei EZ Lysis Buffer, samples were incubated at RT for 5 min. Homogenized tissues were then centrifuged at 500g for 5 min. After removing the supernatant, nuclei were resuspended in 4 mL of Nuclei Suspension Buffer (PBS with 100 μg/mL of BSA) and centrifuged at 500g for an additional 5 min. The resulting pellet was then resuspended in 1 mL of Nuclei Suspension Buffer for FACS. Nuclei were strained through Round-Bottom Polystyrene Test Tubes with a 35 μm Cell Strainer Snap Cap (Falcon) and subjected to FACS using a BD FACSAria II Cell Sorter (Roy J. Carver Biotechnology Center Flow Cytometry Facility, University of Illinois Urbana-Champaign, Urbana, IL). Nuclei were collected in 350 μL of RNeasy Plus Kit Lysis Buffer (Qiagen) and at least 15,000 nuclei were collected for each sample.
5
Isolation of Kidney Mitochondria from Mice
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Mice were sacrificed by dislocation of the cervical spine followed by decapitation. The kidneys were quickly removed and placed in an isolation buffer consisting of 225 mM mannitol (Sigma–Aldrich, St. Louis, MO, USA), 75 mM sucrose (Dia–M, Moscow, Russia), 5 mM Hepes (BioClot, Aidenbach, Germany) (pH = 7.4), and 1 mM ethyleneglycoltetraacetic acid (EGTA) (Sigma–Aldrich, St. Louis, MO, USA), and supplemented with 2 mg/mL fatty acid-free bovine serum albumin (BSA) (Dia–M, Moscow, Russia). The wash buffer used in the centrifugation step had the same composition, except for the addition of BSA.
The kidneys were homogenized using a KIMBLE Dounce tissue grinder (Sigma–Aldrich, St. Louis, MO, USA). The resulting homogenate was centrifuged using a Z36 HK centrifuge (Hermle Labortechnik, Wehingen, Germany) for 5 min at 900× g. The supernatant was transferred into clean tubes and centrifuged for 10 min at 9000× g. Afterwards, the supernatant was removed, and the pellet was resuspended in the wash buffer and centrifuged for 10 min at 9000× g. The resulting mitochondrial pellet was resuspended in the wash buffer.
The kidneys were homogenized using a KIMBLE Dounce tissue grinder (Sigma–Aldrich, St. Louis, MO, USA). The resulting homogenate was centrifuged using a Z36 HK centrifuge (Hermle Labortechnik, Wehingen, Germany) for 5 min at 900× g. The supernatant was transferred into clean tubes and centrifuged for 10 min at 9000× g. Afterwards, the supernatant was removed, and the pellet was resuspended in the wash buffer and centrifuged for 10 min at 9000× g. The resulting mitochondrial pellet was resuspended in the wash buffer.
6
Isolation of Mitochondria from Mouse Brain
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Mitochondria were isolated from fresh brain tissue from both WT and NR2F1 heterozygous (Nr2f1-HET) mice with a Mitochondria Isolation Kit for tissue (ab110168, Abcam, Cambridge, MA, USA), according to the manufacturer's instructions. Briefly, the mouse brain tissue was washed twice with the washing buffer and then suspended in mitochondria isolation buffer to be homogenized with a 2 ml Kimble dounce tissue grinder (D8938-1SET, Sigma-Aldrich) and spun at 1000 g for 10 min at 4°C; the supernatant was then transferred to a new tube and centrifuged again at 12,000 g for 15 min at 4°C. Mitochondria pellets were resuspended twice using mitochondria isolation buffer containing protease inhibitor cocktail (Halt Protease Inhibitor Single-Use Cocktail 100X; 78430, Thermo Fisher Scientific). Final mitochondria isolates were then aliquoted, stored at −80°C and subsequently used for WB analysis.
7
Nuclei Isolation from Muscle Tissue
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Nuclei were isolated from tissue as previously described.72 (link) Briefly, harvested muscle was first homogenized in 2 mL of Nuclei EZ Lysis Buffer (Sigma-Aldrich) using the KIMBLE Dounce Tissue Grinder (Sigma-Aldrich) per the manufacturer’s instructions. After the addition of an extra 2 mL of Nuclei EZ Lysis Buffer to each homogenized tissue, samples were incubated at room temperature (RT) for 5 min. Homogenized tissues were then centrifuged at 500×g for 5 min. After removing the supernatant, nuclei were resuspended in 4 mL of Nuclei Suspension Buffer (PBS with 100 μg/mL BSA and 3.33 μM Vibrant Dye Cycle Violet Stain; ThermoFisher Scientific). Nuclei were centrifuged at 500×g for 1 min and resuspended in 1 mL of Nuclei Suspension Buffer for FACS.
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