Porcine, bovine or commercial BHV portions were prepared from frozen or refrigerated tissue samples, respectively. Tissues were thawed and weighed. Samples (~16–35 mg) were then finely sliced and dissolved in 1 ml TRIS-HCl buffer (50 mM pH5.5) with 10 mM of Ca2+. The solution was thoroughly vortexed for 30 seconds and then 2 mg/ml of collagenase type 2 (Sigma) was added and the mixture was incubated at 37 °C for 1 hour while shaking at 220 rpm. Samples were then put on ice and sonicated with a probe sonicator (Sonic dismembrator, Fisher scientific) three times at a medium power, each for 10 seconds with 30-second intervals incubation on ice. Sonicated solutions together with non-homogenized tissue were then inserted to a glass dounce tissue grinder (2 ml; Sigma) and homogenized with a loose pestle then with a tight pestle (10 times each). Of note, the fresh tissues were fully disrupted while BHV samples were more sturdy. The protein content in the homogenate was evaluated by a standard BCA assay according to manufactures’ protocol (Pierce). The homogenate was kept at −20 °C until use.
Glass dounce tissue grinder
Manufactured by Merck Group
Sourced in United States
The Glass Dounce tissue grinder is a laboratory instrument used for the mechanical disruption and homogenization of biological samples, such as cells and tissues. It consists of a glass vessel with a tight-fitting ground glass pestle that is used to grind and rupture the sample, releasing its cellular contents. The Glass Dounce tissue grinder is a versatile tool commonly used in biochemistry, cell biology, and molecular biology research applications.
Automatically generated - may contain errors
Lab products found in correlation
Rnase inhibitor, by Takara Bio (3 mentions)
35 μm cell strainer, by Corning (3 mentions)
Sonic dismembrator, by Thermo Fisher Scientific (2 mentions)
Collagenase type 2, by Merck Group (1 mentions)
Am2618, by New England Biolabs (1 mentions)
Rnase inhibitor, by New England Biolabs (1 mentions)
Macs smartstrainer, by Miltenyi Biotec (1 mentions)
Lysis buffer, by Miltenyi Biotec (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Mitochondria isolation kit, by Miltenyi Biotec (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Pepstatin a, by Research Products International (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Ez prep, by Merck Group (1 mentions)
Nuc 101, by Merck Group (1 mentions)
Facsaria 3 flow cytometer, by BD (1 mentions)
Bz x710 microscope, by Keyence (1 mentions)
9 protocols using glass dounce tissue grinder
1
Extraction and Characterization of Bovine or Porcine BHV
2
Protein Extraction from French Food Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
French food samples (Additional file 1 : Table S1) were shipped frozen from France to Tel Aviv University and stored at − 80 °C. Samples were thawed, 50 mg of each food sample was weighed, incubated at − 80 °C for 2 h, then lyophilized for overnight. Dried samples were dissolved in 1 ml of lysis buffer (50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), thoroughly vortexed for 30 s, put on ice, then sonicated with a probe sonicator (Sonic Dismembrator, Fisher Scientific) three times at a medium power, each for 10 s with 30-s intervals incubation on ice. Sonicated solutions were then inserted into a glass Dounce tissue grinder (2 ml; Sigma) and homogenized with a loose pestle then with a tight pestle (10 times each). The homogenate was centrifuged 10,000×g for 5 min to remove pelleted nuclei and cell debris, and protein content in the supernatant homogenate was evaluated by a standard BCA assay according to manufacturer’s protocol (Pierce). The homogenate was stored at − 20 °C until use.
3
Endolysosomal Proteolytic Stability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteolytic stability to endolysosomal proteases was investigated using the endolysosomal degradation assay. Endolysosomes were isolated from murine JAWS II dendritic cells by differential centrifugation as previously described [18 (link)]. Briefly, 5 × 107 cells were resuspended in 10 mM Tris/acetate pH 7.0 containing 250 mM sucrose and homogenized on ice using a glass Dounce tissue grinder (Sigma-Aldrich, St. Louis, MO, USA). The cell homogenate was centrifuged for 10 min at 6000× g to obtain a post-nuclear supernatant. After an ultracentrifugation step for 60 min at 100,000× g, the microsomal content was released by five freeze/thaw cycles on liquid nitrogen and room temperature, respectively, and subsequently stored at −20 °C. Allergens (5 µg) were incubated with 7.5 µg of isolated microsomal proteins in a final volume of 20 µL containing 100 mM citrate buffer pH 4.8 and 2 mM dithiothreitol; the proteolytic digestion at 37 °C was monitored up to 72 h. Reactions were stopped by denaturation at 95 °C for 5 min at the time points of 0, 0.5, 1, 3, 8, 16, 24, 48 and 72 h, and samples were stored at −20 °C until further use.
4
Isolation of Intact Nuclei from Mouse Hearts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice hearts were harvested and frozen after sacrificing them. Frozen tissue samples were cut into pieces <0.5 cm and homogenized using a glass Dounce tissue grinder (Sigma, cat. no. D8938). The tissue was homogenized 25 times with pestle A and 25 times with pestle B in 2 mL of ice-cold nuclei EZ lysis buffer. The sample was then incubated in ice for 5 min with 3 mL of cold EZ lysis buffer. Nuclei were centrifuged at 500 g for 5 min at 4°C, washed with 5 mL ice-cold EZ lysis buffer, and incubated on ice for 5 min. After centrifugation, the nuclear pellet was washed with 5 mL nuclei suspension buffer [NSB; consisting of 1% PBS, 0.01% BSA, and 0.1% RNase inhibitor (Clontech, cat. no. 2313A)]. Isolated nuclei were resuspended in 2 mL NSB, filtered through a 35 μm cell strainer (Corning-Falcon, cat. no. 352235) and counted. A final concentration of 1,000 nuclei per μl was used for loading onto a 10x channel.
5
Single-nucleus RNA-seq of tissue samples
6
Nuclei Isolation from Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nucleus isolation was done as previously described8 (link). Briefly, tissue samples were cut into pieces <0.5 cm and homogenized using a glass Dounce tissue grinder (Sigma, cat. no. D8938). The tissue was homogenized 25 times with pestle A and 25 times with pestle B in 2 ml of ice-cold nuclei EZ lysis buffer. The sample was then incubated on ice for 5 min, with an additional 3 ml of cold EZ lysis buffer. Nuclei were centrifuged at 500g for 5 min at 4 °C, washed with 5 ml ice-cold EZ lysis buffer and incubated on ice for 5 min. After centrifugation, the nucleus pellet was washed with 5 ml nuclei suspension buffer (NSB; consisting of 1× PBS, 0.01% BSA and 0.1% RNase inhibitor (Clontech, cat. no. 2313A)). Isolated nuclei were resuspended in 2 ml NSB, filtered through a 35 μm cell strainer (Corning-Falcon, cat. no. 352235) and counted. A final concentration of 1,000 nuclei per µl was used for loading on a 10x channel.
7
Isolation and Lysis of Mitochondria
8
Isolation of Mouse Brain Nuclei
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were anesthetized with 3% isoflurane and brains were removed and placed into ice-cold oxygenated artificial cerebrospinal fluid (aCSF). SC were dissected from the midbrain and placed into RNAlater (Invitrogen, AM7021) and stored at 4°C overnight. To ensure the quality of the experiment, two replicates were conducted and five mice were used for each replicate. On the day for the experiment, tissue samples were washed with phosphate buffered saline (PBS) (Gibco, REF 10010–023) and cut into pieces <1 mm and were homogenized using a glass Dounce tissue grinder (Sigma, Cat# D8938) in 2 ml of ice-cold EZ PREP (Sigma, Cat# NUC-101). Then the nuclei suspension was transferred into a 15 ml tube and incubated on ice for 5 min with 2 ml of ice-cold EZ PREP added. After incubation, the nuclei were centrifuged at 500× g for 5 min at 4°C. The nuclei were re-suspended with 4 ml ice-cold EZ PREP and incubated on ice for another 5 min. Then the nuclei were centrifuged at 500× g for 5 min at 4°C and washed in 4 ml Nuclei Suspension Buffer (NSB; consisting of 1× PBS, 0.04% BSA, and 0.1% RNase inhibitor [Clontech, Cat# 2313A]). After being re-suspended in 2 ml NSB, the nuclei were filtered with a 35 μm cell strainer (Corning, Cat# 352235). The nuclei density was adjusted to 1,000,000 nuclei/ml and placed on ice for use.
9
Isolation and FACS Sorting of Murine Hippocampal Nuclei
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse hippocampi were harvested from male mice at the age of 250 days and immediately snap frozen in liquid nitrogen and stored at -80°C until further processed for nuclei isolation. Nuclei were isolated from a single mouse hippocampus in 2 ml of pre-chilled EZ PREP lysis buffer (NUC-101, Sigma) using a glass Dounce tissue grinder (D8938, Sigma) (25 strokes with pastel A and 25 strokes with pastel B) followed by incubation for 5 minutes on ice with additional 2 ml of EZ PREP buffer. During incubation, 1 µM DAPI was added to the homogenate. The homogenate was then filtered through a 30 µM FACS tube filter. A BD FACSAria III Flow Cytometer with a 70 µm nozzle configuration was used to sort the nuclei based on the fluorescent DAPI signal at 4°C. As CNS nuclei vary strongly in size, no doublet discrimination was performed based on FSC or SSC to avoid bias against nucleus size. Nuclei were then counted based on brightfield image and DAPI fluorescence using a Neubauer counting chamber and a Keyence BZX-710 microscope.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!