Total protein extracts were prepared from tail biopsies minced in 150 μL Tris–HCl, pH 7.8, and subjected to sonication and freeze-thawing cycles. For protein extracts from total bone, the tissue was frozen in liquid nitrogen and homogenized using aluminum beads and the TissueLyser II instrument (Qiagen, Hilden, Germany). Tissue debris were removed by centrifugation at 4°C. Protein concentration in the supernatants were estimated using the DC™ Protein Assay Kit II (Bio-Rad, Berkeley, CA, USA) following the manufacturer’s instructions, on a Synergy™ H4 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). DPP3 enzymatic activity was performed as reported,(31 (link)) with minor modifications. Briefly, 50 to 80 μg of protein extracts were assayed with 0.04 mM Arg-Arg-β-naphthylamide (Sigma-Aldrich, Saint Louis, MO, USA) in 250 μL Tris–HCl, pH 8.5, at room temperature (RT). The reaction was stopped by adding 50 μL of 2 M acetate buffer, pH 4.5, containing 10% Tween and 1.5 mg/mL Fast Blue B Salt (all chemicals from Sigma-Aldrich). The absorbance of the released product (β-naphthylamine) was measured at 530 nm, using SynergyTM H4 Microplate Reader. The enzymatic activity was expressed as percentage of the wild-type (WT) value.
Tissuelyser 2 instrument
Manufactured by Qiagen
Sourced in Germany
The TissueLyser II is a high-throughput, parallel homogenization instrument designed for efficient disruption and lysis of a wide range of sample types, including plant, animal, microbial, and human tissues. The instrument uses bead-beating technology to disrupt samples and release nucleic acids, proteins, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Rneasy plus kit, by Qiagen (2 mentions)
Rnalater solution, by Merck Group (2 mentions)
Rlt plus buffer, by Qiagen (2 mentions)
Reagent dx antifoam solution, by Qiagen (2 mentions)
Protoscript 2 enzyme, by New England Biolabs (2 mentions)
Dc protein assay kit 2, by Bio-Rad (1 mentions)
Fast blue b salt, by Merck Group (1 mentions)
Arg arg β naphthylamide, by Merck Group (1 mentions)
Synergy h4 microplate reader, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Multiscribe reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit with rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Viia7 real time pcr detection system, by Thermo Fisher Scientific (1 mentions)
Ssoadvanced sybr green supermix, by Bio-Rad (1 mentions)
Purezol reagent, by Bio-Rad (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
14 protocols using tissuelyser 2 instrument
1
Protein Extraction and DPP3 Activity Assay
2
Evaluating SARS-CoV-2 Infection in Hamsters
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On day 21 after booster injection, hamsters were infected with WT SARS-CoV-2 at a dose of 1 × 104 TCID50 intranasally under intraperitoneal ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia. Virus material was diluted in DMEM medium and injected 100 µl into the nose (approximately 50 µl in each nostril) using a pipette. Animals were observed for 7 days twice daily after challenge and body weight recorded daily. On Days 3 and 7 after infection, half of the animals (3/6) from each group were euthanized and collected nasal turbinate and lung samples. Three lobes of the right lung from each animal were fixed in 10% formalin for histopathological examination. Two lobes of the left lung were homogenized in 1 ml DMEM using a TissueLyser II instrument (QIAGEN) at 300 vibrations/min for 60 s, supernatant collected after centrifugation (5000 g for 15 min at 4 °C) was stored at -70 °C for detection of viral RNA and infectious viral titer.
3
Intranasal SARS-CoV-2 Vaccine Evaluation in Hamsters
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Six- to eight-week-old male Syrian hamsters obtained from the NSCEDI Laboratory Animal Breeding Facility were used in the vaccine trial. Animals were placed in ventilated cages with HEPA filters for 7 days prior to the experiment for acclimatization. Hamsters were immunized with NP-vaccine formulations (NP vaccine and NP-CpG vaccine, n = 6/group) containing RBD protein 5 µg/dose or PBS (control group, n = 6) intranasally in a 100 µL volume under ketamine (80 mg/kg) and xylazine (8 mg/kg) anesthesia twice at 21-day intervals. For comparison, an RBD-alum vaccine containing 5 µg/100 µL of RBD protein was administered intramuscularly.
At day 21 after booster vaccination, hamsters were intranasally challenged with SARS-CoV-2 at a dose of 1 × 104 TCID50/100 µL under ketamine-xylazine anesthesia and observed for 7 days post infection with daily measurement of body weight. On days 3 and 7 post challenge, half of the animals (3/6) from each group were euthanized and nasal turbinates and lung samples collected. Three lobes of the right lung from each animal were fixed in 10% formaldehyde for histopathological studies. Two lobes of the left lung were homogenized in 1 mL DMEM using a TissueLyser II instrument (QIAGEN) at 300 vibrations/min for 60 s, the supernatant after centrifugation (5000 g for 15 min at 4 °C) was collected and stored at − 70 °C for later determining the viral titer.
At day 21 after booster vaccination, hamsters were intranasally challenged with SARS-CoV-2 at a dose of 1 × 104 TCID50/100 µL under ketamine-xylazine anesthesia and observed for 7 days post infection with daily measurement of body weight. On days 3 and 7 post challenge, half of the animals (3/6) from each group were euthanized and nasal turbinates and lung samples collected. Three lobes of the right lung from each animal were fixed in 10% formaldehyde for histopathological studies. Two lobes of the left lung were homogenized in 1 mL DMEM using a TissueLyser II instrument (QIAGEN) at 300 vibrations/min for 60 s, the supernatant after centrifugation (5000 g for 15 min at 4 °C) was collected and stored at − 70 °C for later determining the viral titer.
4
Tissue Preparation for Gene Expression
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Tissue samples for each set of experiments were obtained and processed in parallel for both animal groups, and were performed at the same time of the day (early in the morning). The euthanasia and tissue collection for gene expression analysis were set 60 min after the high acoustic stimulation as established previously by López-López et al. (2017) (link). For tissue sampling, all animals were deeply anesthetized under gas anesthesia (2.5% isoflurane), the brains were removed quickly after decapitation and the IC was isolated, surgically removed, and placed in QIAzol Lysis Reagent (#79306, QIAgen). Then, the samples containing the IC tissue were homogenized using TissueLyser II instrument (#85300, QIAgen), according to the manufacturer’s instructions. After separating the different phases, the aqueous phase was used to extract RNA following the instructions of the RNeasy Mini Kit (#74104, QIAgen). RNA concentrations and quality were assessed using an Agilent 2100 Bioanalyzer to assess the integrity of the 18S and 28S rRNA bands, as well as an RNA integrity number (RIN) > 8.0, with 0 corresponding to fully degraded RNA and 10 corresponding to intact RNA, were used for data analysis (López-López et al., 2017 (link)).
5
Isolation and Purification of RNA from Aortic Tissue
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Tissue samples stabilized in RNAlater solution (Sigma) were homogenized using a TissueLyser II instrument (Qiagen), in the presence of Reagent DX antifoam solution and RLT Plus buffer (Qiagen). Total RNA was purified using the RNeasy Plus kit (Qiagen). Reverse transcription was carried out using ProtoScript II enzyme (New England Biolabs) and random 15-mer primers. The resulting cDNA samples were purified using the QIAquick PCR purification kit (Qiagen). To isolate RNA separately from the endothelial and medial layers of mouse thoracic aorta, vessels were flushed with Qiazol reagent, as described previously [43 (link)]. Total RNA was purified using the miRNeasy kit (Qiagen) from both fractions. Reverse transcription was carried out using random primers and the Multiscribe reverse transcriptase provided in the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems).
6
Quantitative PCR Analysis of Osteoblast Gene Expression
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Total RNA was extracted from primary osteoblast cell cultures using the PureZOL™ Reagent (Bio-Rad), following the manufacturer’s instructions. For RNA extraction from murine flushed long bones (n = 4 WT, 7 KO), the frozen tissue was crashed in a mortar, then transferred in tubes and homogenized in a TissueLyser II instrument (Qiagen) in the presence of PureZOL™ Reagent; afterwards, standard procedures were applied. Reverse transcription was carried out using 1.0 μg total RNA and High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative PCR (qPCR) was performed using SsoAdvanced™ SYBR® Green Supermix (Bio-Rad) and gene-specific primers as detailed in Table 1 . The amplification was performed using the ViiA7 Real-Time PCR Detection System (Applied Biosystems) with the following cycling conditions: cDNA denaturation and polymerase activation step at 95°C for 20 s followed by 40 cycles of denaturation at 95°C for 1 s and annealing at 60°C for 20 s; extension step for 60 cycles at 65°C for 30 s and melting curve analysis step at 65°C–95°C with 0.5°C increment for 2 s/step. The relative gene expression analysis of target genes was conducted following the comparative 2−ΔΔCT method and the normalized expression was calculated as arbitrary units (AU) in comparison with pertinent controls.
7
Protein Extraction and Visualization
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The pellet was resuspended in 500 μL yeast suspension buffer (YSB) containing protease inhibitor cocktail. Cells were disrupted with a TissueLyser II instrument (Qiagen, Germany) and centrifuged at 12,000 rpm. The pellet was resuspended in YSB and reextracted as described above. The supernatant was centrifuged at 22,000 × g for 70 min at 4°C. Then, 15 μL of YSB was used to resuspend the new pellet. After being mixed with an equal amount of SB, the mixture was incubated at 37°C for 5 min and loaded on a 10% Tris Glycine gel (Invitrogen, Waltham, MA, USA) for SDS-PAGE analysis. The gel was visualized using an LAS-1000 luminescent image analyzer (Fujifilm, Tokyo, Japan).
8
Mouse Gene Expression Microarray Protocol
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Total RNA was extracted using the mirVanaTM kit (Life Technologies, ThermoFisher Scientific). Tissue disruption was accomplished with a TissueLyser II instrument (Qiagen, Carlsbad, CA). Sample concentration was determined using the NanoDrop® ND-1000, and RNA integrity was analyzed using the Agilent 2100 Bioanalyzer Lab-On-A-Chip Agilent 6000 Series II chip. Sample labeling and hybridization was performed per the manufacturer’s protocol. Samples were hybridized to the SurePrint G3 Mouse GE 8x60K Microarray and scanned using the Agilent G2505C Microarray Scanner. Raw data were quality-assessed, filtered for outliers and normalized to remove non-biological variation. A 10% false discovery rate and p-value adjustment using the Bioconductorlimma package yielded significantly differentially expressed genes (Core Facility, Nationwide Children’s, Columbus, OH). Microarray data may be accessed at Gene Expression Omnibus (www.ncbi.nlm.nih.gov/GEO ), using accession number GSE72007.
9
Culicoides Homogenization for Virus Analysis
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Homogenates of individual heads were used to investigate virus dissemination and thoraxes for species identification (described above). Briefly, heads were removed by using sterile needles (Fine-Ject, Tuttingen, Germany) and transferred into 1.5 ml Eppendorf tubes containing 100 μl of GMEM growth media (see above). For homogenization, Culicoides were either manually processed for 30 s using sterilised polypropylene pestles (Sigma-Aldrich, Gillingham, Dorset, UK) mounted to a motorized grinder (Micro Handrührer, Carl Roth, Karlsruhe, Germany) or homogenized for 2 × 30 s with 3 mm diameter stainless steel ball bearings using the Tissue Lyser II Instrument (Qiagen) as previously described [47 (link)]. After homogenization, each sample was filled up with 900 μl of Glasgow MEM giving a final volume of 1 ml/homogenate. All tubes were then vortexed and centrifuged for 5 m at 13000× g. Aliquots were immediately processed for viral RNA extraction and the rest of the homogenates was kept at 4 °C.
10
Fecal DNA Extraction Protocol
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Genomic DNA was extracted from ~100 mg of feces with the use of a Nucleospin DNA Stool Kit (740472, Macherey-Nagel) according to the manufacturer’s instructions with the following modifications; for sample preparation each Nucleospin Tube Type A containing 100 mg of feces was loaded into pre-chilled adapters and shaken twice for 30 sec with a TissueLyser II instrument (Qiagen) at a frequency of 25 Hz. Following lysis at 70°C for 5 mins, the samples were further disrupted by vigorous shaking with a ThermoMixer (Eppendorf) at room-temperature, 2000 rpm for 10 min. Nucleic acids were eluted in 50 μl elution buffer and stored at -20°C.
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