Hippocampal samples were weighed and extracted using a 2-step extraction protocol with modifications as described previously (Youmans et al., 2012 (link); Tai et al., 2013 (link), 2014a (link); Thomas et al., 2016 (link), 2017 (link); Marottoli et al., 2017 (link), 2019 (link)), in order to separate out soluble and detergent-soluble proteins. Briefly, samples were homogenized using a bead mill (Fisherbrand) at 6 m/s for 1 cycle of 30 s in ice cold TBS at 10 μl/mg of brain tissue, centrifuged (100,000 × g for 30 min), and aliquoted. The resulting pellet was then resuspended in SDS buffer (1% SDS + 10 mM NaF + 2 mM Na3VO4 + 1× protease inhibitor cocktail in 20 mM HEPES; pH = 7.4), mixed via end-over-end rotation for 30 min at 4°C, sonicated (20% amplification, 3 cycles), centrifuged (100,000 × g for 30 min), and aliquoted. TBS and SDS buffer aliquots were flash frozen in liquid nitrogen and stored at −80°C. Total protein was quantified in TBS and SDS buffer extracts using the Pierce BCA Protein Assay Kit.
Bead mill
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
The Bead mill is a lab equipment used for efficient homogenization and disruption of biological samples. It utilizes a high-speed rotating motion to agitate samples along with beads, resulting in effective cell lysis and tissue disruption.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorimeter, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (1 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Dnase 1, by Qiagen (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Dnase, by Bio-Rad (1 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Bio-Rad (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
P450 glo, by Promega (1 mentions)
Lysing matrix d, by MP Biomedicals (1 mentions)
Jetpei, by Polyplus Transfection (1 mentions)
14 protocols using bead mill
1
Hippocampal Protein Extraction Protocol
2
Rectal Tissue Homogenization for RT-QuIC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rectal biopsy samples were weighed and placed in homogenate tubes containing 1.0 mm zirconia/silica beads. Phosphate Buffered Saline (1X PBS) was added to create 10% (w/v) homogenate (i.e., 0.1 g tissue plus 1 mL 1X PBS) and a ¼ inch ceramic bead was added to each tube. Samples were homogenized in a BeadMill tissue homogenizer (Fisher) at Power 6, 1 min on, 5-minute rest with 3 replicates at 4°C. The resulting homogenate was stored at -80°C until used to seed RT-QuIC reactions.
3
RNA Extraction from Fish Gill Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following the removal of the whole, 2nd left gill from RNALater, Total RNA was extracted using the RNeasy Mini Plus Kit (Qiagen, Germany) according to the manufacturer’s instructions. Briefly, 30 mg gill tissue from each individual fish gill, irrespective of the presence of absence of mucoid lesions, was homogenized using a bead mill (Fisher Scientific) and 2.8 mm ceramic beads in 350 µl RLT lysis buffer and 1 µl DX antifoam reagent (Qiagen, Germany), 5 pulses/sec, 10 s, repeated 3 times. The optional DNase I (Qiagen, Germany) step was included to ensure complete elimination of gill genomic DNA. RNA was eluted in 70 µL of nuclease -free water and stored at − 80 °C until required. RNA was quantified using the Qubit RNA Assay Kit in Qubit 2.0 Fluorimeter (Thermo Fisher Scientific, USA). RNA integrity (RIN) was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies CA, USA). Total RNA with RIN ≥ 8.0 or higher were used for library construction.
4
Gene Expression Analysis by qRT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression was assessed on day 3 using qRT-PCR. Tissues were lysed in buffer RLT (Qiagen), homogenized using a Bead Mill (Fisher Scientific), then centrifuged at 20,000× g for 3 min. The supernatant was then collected and stored at − 80 °C until RNA was extracted. An RNeasy Kit (Qiagen) was used for RNA isolation. RNA quality and concentration were checked with a Nanodrop One Spectrophotometer (ThermoFisher). Following DNAse treatment (BioRad) and reverse transcription (T100 Thermal Cycler, BioRad) PCR reactions were prepared, using the following primers: Prdx5 (peroxiredoxin-5, BioRad), Nfe2l2 (nuclear factor erythroid 2-related factor 2, also known as NRF2, BioRad), and VEGFa (vascular endothelial growth factor A, BioRad). The housekeeper GAPDH (BioRad) was used as the internal control gene. PCR reactions were run using a CFX96 Real-Time System (BioRad), and results processed with BioRad CFX Maestro software.
5
Cytochrome P450 Activity Assay in Cochlea and Liver
6
Blood and Brain Tissue Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were anesthetized via intraperitoneal injection with ketamine (100 mg/kg) and xylazine (10 mg/kg). For plasma isolation, blood was drawn by cardiac puncture of the right ventricle followed by centrifugation at 1500g for 20 minutes at 4 °C. Plasma was flash frozen in liquid nitrogen and stored at −80 °C. Following blood draw, transcardial perfusion was performed with ice-cold TBS at a rate of 4 mL/min for 4 minutes. For immunohistochemical analysis, left hemi brains were frozen in OCT and stored at −80 °C until processing. For biochemical analysis, right hemicortices were isolated and homogenized using a bead mill (Fisherbrand) at 6 m/s for 1 cycle of 30 s in SDS lysis buffer (1% SDS+10 mmol/L NaF+1× HALT protease inhibitor cocktail in 20 mmol/L HEPES; pH 7.4). Samples were then centrifuged at 500g for 5 minutes at 4 °C, sonicated (20% amplification, 3 cycles), and then centrifuged again (25 000g for 20 minutes at 4 °C). Aliquots of the resulting supernatant were then flash frozen in liquid nitrogen and stored at −80 °C. Total protein was quantified using the Pierce BCA Protein Assay Kit. Brains from a separate cohort of mice were drop-fixed overnight in 4% paraformaldehyde (PFA), cryopreserved in 30% sucrose, sectioned, and then stored at 4 °C in cryoprotectant until processing.
7
RNA-Mediated Antiviral Immune Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro transcribed VSV 5′-triphosphate PAMP RNA was complexed with in vivo JetPEI (PolyPlus) using an N/P (nitrogen/phosphate) ratio of 8. Each batch of in vitro transcribed PAMP RNA was screened in cell culture to ascertain its ability to elicit an interferon response (Supplemental Fig. S8C ). RNA lacking immunostimulatory activity was used in the mock transfected mice. No transfection reagent was used in the untransfected mice. Treated mice were administered with 15 μg of RNA through the intraperitoneal (IP) route. For VSV infection, mice were infected with wild-type VSV Indiana strain (2 × 106 PFU) via intravenous tail vein injection. Tissue samples and sera were collected 6 h postadministration. Harvested tissue samples were immediately transferred to lysing matrix tubes (Lysing Matrix D, MP Biomedicals) containing TRIzol and were homogenized in a homogenizer (Bead Mill, Fisher Scientific) before snap-freezing in liquid nitrogen.
8
Brain Tissue Homogenization and Preservation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen tissue was obtained, with the contralateral half being fixed and used for histopathology as described above. Multiple brain sections were dissected from the samples of mid-frontal lobe tissue to ensure representative samplings. 10% w/v brain tissue homogenates were prepared in ice-cold 1 × PBS with cOmplete protease inhibitors, EDTA-free (Roche) and homogenized using 1 mm zirconia/silica beads (BioSpec Products) in a BeadMill (Fisherbrand). Homogenates were placed on ice for 5 min before centrifugation at 2000×g for 2 min. Supernatant was collected, aliquoted and stored at − 80 °C.
9
Drosophila RNA-seq Library Prep
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare libraries for RNA sequencing we collected replicates of 30 mated flies, sexes separately, of up to two lines per genotype between 8:00 AM and 11:00 AM and flash froze them on dry ice in 2-ml tough microtubes (Thermofisher, Waltham, MA). Total RNA was extracted using a modified version of the RNAeasy plus mini kit (Qiagen, Hilden, Germany) protocol. Four 2.4-mm metal beads (Thermofisher) were added to each sample tube and the flies were homogenized in a bead mill (Thermofisher) for 2 min at 5 m/s, after which the RNA was eluted with a total of 30 μl H2O. We used the NuQuant +UDI, Drosophila AnyDeplete kit (Tecan, Männedorf, Switzerland) to deplete ribosomal RNA and prepare bar-coded cDNA libraries for sequencing after 17 cycles of amplification. We used the Qubit 1X HS dsDNA HS kit (Thermofisher) to quantify the libraries and high-sensitivity D1000 screentape (Agilent, Santa Clara, CA) to check the quality of size selection. We then normalized the libraries to 5 nM concentration and pooled them in order to get a final equimolar concentration of 3 nM. The pooled libraries were run on an S1 flow cell on the Illumina Novaseq6000 platform (Illumina, San Diego, CA).
10
Extraction and Purification of Cortical RNA
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!