Primary hippocampal neuronal cultures were treated at 5 days in vitro (DIV) with respective chemicals for 4 days unless other duration of treatment is indicated. Following the treatment, cultures were washed with cold PBS (pH 7.2), lysed in cold lysis buffer (N-PER, Thermo Scientific, MA)and then harvested with a cell scraper, followed by centrifugation at 10,000 × g for 10 min. Protein concentrations were determined via BCA Assay (Pierce Biotechnology, IL). For tissue protein extraction, 30 mg of hippocampal, cortical and hypothalamic tissue samples were homogenized using a Bullet Blender 24 Homogenizer (Next Advance, NY) in T-PER (Pierce Biotechnology, IL) supplemented with protease and phosphatase inhibitors (Roche Applied Science, IN) and 100 μL 0.5 mm glass beads (Next Advance, NY) at speed 8 for 3 min at 4°C followed by centrifugation at 12,000 rpm for 8 min at 4°C. Supernatants were transferred to new microcentrifuge tubes and protein concentrations were determined via BCA Assay (Pierce Biotechnology, IL).
Bullet blender 24 homogenizer
Manufactured by Next Advance
The Bullet Blender 24 Homogenizer is a lab equipment designed for efficient homogenization of samples. It operates using a high-speed rotating blade to thoroughly mix and blend various materials in a sample container.
Automatically generated - may contain errors
Lab products found in correlation
T per, by Thermo Fisher Scientific (2 mentions)
Truseq rna sample preparation kit v2, by Illumina (2 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
N per, by Thermo Fisher Scientific (1 mentions)
0.5 mm glass beads, by Next Advance (1 mentions)
2200 tapestation system, by Agilent Technologies (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Hiscansq system, by Illumina (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Agencourt rnaclean xp, by Beckman Coulter (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Paxgene blood rna kit, by Preanalytix (1 mentions)
Lysing matrix d tube, by MP Biomedicals (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using bullet blender 24 homogenizer
1
Primary Hippocampal Neuron Culture Protocol
2
Tissue Homogenization for Protein Extraction
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30 mg of tissue from indicated brain and peripheral organs was homogenized using the Bullet Blender 24 Homogenizer (Next Advance, Averill Park, NY) with 100 µL 0.5 mm glass beads in variable volumes of tissue protein extraction reagent (TPER, Life Technologies Cat. #78510) supplemented with protease and phosphatase inhibitors according to the manufacturer’s instructions.
3
RNA Sequencing Library Preparation
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RNA was isolated using TRI Reagent (Applied Biosystems) according to the
manufacturer’s protocol. Muscle samples were homogenized using a Bullet Blender
24 homogenizer (Next Advance). The RNA (Agencourt RNAClean XP kit) was purified
by a bead method and its quality and quantity were assessed fluoremetrically
(Qubit Fluorometer, Invitrogen) and by the TapeStation 2200 system (RNA tapes,
Agilent). RNA integrity number (RIN) was in the range between 6.8-8. Ribosomal
RNA from 5000 ng of total RNA was removed using a Ribo-Zero Gold rRNA Removal
Kit (Human/Mouse/Rat) (Epicentre). The absence of rRNA was verified on the
TapeStation 2200 system. The elimination of rRNA leads to a flattening of the
differences in transcript levels between the groups and allows for sequencing of
cDNA libraries presenting low frequency (Benes
et al., 2011 ). A TruSeq RNA Sample Preparation
Kit v2 (Illumina) was used to prepare cDNA libraries from 100 ng aliquots of
rRNA-depleted samples according to the manufacturer’s protocol. The cDNA samples
were ligated with indexed adaptors in the order shown in
Table
S1 . The libraries were amplified in 15
cycles of PCR, and their quantity was estimated using the Qubit 2.0 Fluorometer
and 2200 TapeStation (D1000 tapes). The final concentration of the cDNA
libraries was normalized to 10 nM, after which the libraries were pooled
(Table
S1 ).
manufacturer’s protocol. Muscle samples were homogenized using a Bullet Blender
24 homogenizer (Next Advance). The RNA (Agencourt RNAClean XP kit) was purified
by a bead method and its quality and quantity were assessed fluoremetrically
(Qubit Fluorometer, Invitrogen) and by the TapeStation 2200 system (RNA tapes,
Agilent). RNA integrity number (RIN) was in the range between 6.8-8. Ribosomal
RNA from 5000 ng of total RNA was removed using a Ribo-Zero Gold rRNA Removal
Kit (Human/Mouse/Rat) (Epicentre). The absence of rRNA was verified on the
TapeStation 2200 system. The elimination of rRNA leads to a flattening of the
differences in transcript levels between the groups and allows for sequencing of
cDNA libraries presenting low frequency (
et al., 2011
Kit v2 (Illumina) was used to prepare cDNA libraries from 100 ng aliquots of
rRNA-depleted samples according to the manufacturer’s protocol. The cDNA samples
were ligated with indexed adaptors in the order shown in
S1
cycles of PCR, and their quantity was estimated using the Qubit 2.0 Fluorometer
and 2200 TapeStation (D1000 tapes). The final concentration of the cDNA
libraries was normalized to 10 nM, after which the libraries were pooled
(
S1
4
Chicken Hypothalamus RNA Sequencing
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RNA was isolated with PureLink™ RNA Mini Kit (Ambion) according to manufacturer's recommendations. Hypothalamus samples were homogenized with beads using a Bullet Blender24 homogenizer (Next Advance). RNA was purified by an Agencourt® RNAClean™ XP (Beckman Coulter, Inc.). Quantitative and qualitative evaluation of RNA was performed using a TapeStation 2200 (Agilent). The RIN was in the range 7.3–8.4. The cDNA libraries were prepared for two age groups: 22- and 45-day-old chickens, with a TruSeq RNA Sample Preparation Kit v2 (Illumina) according to the protocol. The libraries were indexed using individual adaptors in the order shown in Table S1 and normalized to 10 nM after which the libraries were pooled. Flow-cell clustering was performed using a TruSeq SR Cluster Kit v3-cBot HS. The sequencing was conducted on a HiScanSQ System (Illumina) in single 101-bp cycles using TruSeq Kit v3-HS chemistry according to Ropka-Molik et al. [14 (link)].
5
Nucleic Acid Extraction from Placenta and Cord Blood
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Cord blood DNA extraction was carried out using the Qiagen Puregene Blood Core Kit C (Qiagen) according to the manufacturer's protocol. Cord blood RNA was extracted using the PAXgene Blood RNA kit (PreAnalytix) according to the manufacturer's protocol. Placental tissue was homogenized by placing 25 mg of sample into Lysing Matrix D tubes (MP Biomedicals) in the Bullet Blender 24 homogenizer (Next Advance), and RNA was extracted from these samples using the Qiagen RNeasy Mini Kit (Qiagen). DNA extraction from placental chorionic villi tissue was performed by washing the tissue several times in 1Â phosphate-buffered saline followed by extraction using the QIAamp DNA Mini Kit (Qiagen) with an RNase A step to remove any RNA contamination. Placental and cord blood RNA samples were run on a 1.5% agarose gel for quality assurance and then converted into cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), with the following thermal profile: 10 minutes at 25 C, 2 hours at 37 C, and hold at 4 C. For placental chorionic villi samples, 1 mg of RNA was used in the conversion reaction, whereas, for the cord blood samples, 0.25 mg of RNA was converted as this tissue had an overall lower concentration of RNA.
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