To assess in vivo efficacy of antibodies, female 6–8 weeks old DBA/2J mice (4–5 per group) were each injected with mAb at a dose of 10 mg/kg (in 100 μL of PBS) intraperitoneally. Two hours post antibody administration, mice were given anaesthesia and the day 0 weight was recorded. Next, each mouse was intranasally infected with 5 mLD50 of virus. Mice were then monitored daily for weight loss. As mentioned above, loss of 25% of initial body weight led to humane euthanization of the mouse and the mouse was scored dead. Weight loss and survival data were analysed in Prism 7. To assess inhibition of virus replication in the lungs, antibodies were used at a concentration of 10 mg/kg. Two hours post administration of each antibody (5–6 mice per group), mice were given anaesthesia and infected with 1 LD50 of virus. A lower virus dose was used to increase sensitivity of this assay. Three mice from each group were sacrificed on day 3 while the remaining three mice were euthanized on day 6. Lungs from each mouse were harvested and homogenized using a BeadBlaster 24 (Benchmark) homogenizer. Lung homogenates were frozen and were then analysed in a standard plaque assay on MDCK cells to assess viral titres.
Beadblaster 24
Manufactured by Benchmark Scientific
Sourced in United States
The BeadBlaster 24 is a high-throughput bead-based homogenizer designed for efficient sample disruption and nucleic acid extraction from a variety of sample types. The device utilizes rapid oscillation of samples containing beads to effectively lyse cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Balb c mice, by Jackson ImmunoResearch (2 mentions)
Addavax, by InvivoGen (2 mentions)
C57bl 6, by Jackson ImmunoResearch (2 mentions)
Rnalater, by Qiagen (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Mic qpcr cycler, by Bio Molecular Systems (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96 instrument, by Roche (1 mentions)
Anti myc, by BioLegend (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
O dianisidine, by Merck Group (1 mentions)
Sensifast probe no rox one step kit, by Meridian Bioscience (1 mentions)
Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Rnase, by New England Biolabs (1 mentions)
Sigma 1 14, by Merck Group (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
31 protocols using beadblaster 24
1
In Vivo Antibody Efficacy Evaluation
2
Placental RNA Extraction and qRT-PCR Analysis
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Total RNA from mouse placentae collected on E15.5, E16.5, and E17.5 was isolated using Trizol Reagent (Ambion, Ref # 15596026) and a bead shaker (BeadBlaster 24, Benchmark Scientific, SKU: D2400) with a minimum of four placentae were pooled per dam. RNA was DNase-treated (Invitrogen, Ref # AM1906) and then reverse-transcribed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Ref # 4368814). Relative transcript abundance for the genes of interest was quantified using PowerSYBR Green PCR Master Mix (Applied Biosystems, Cat # 4367659), the Roche LightCycler 96 Instrument (software version 1.01.01.0050), and the Mic qPCR cycler (Biomolecular systems, firmware version 2.25). Each sample was assayed in duplicate for target and housekeeping genes. Average Ct values of target genes were normalized to average Ct values of Ubc as the reference gene and relative transcript abundance of genes of interest was determined using the ΔΔCt method. Transcript expression in individual mice is presented relative to the mean expression value in UP mice at E6.5. Details of primer sets are summarized in S2 Table .
3
Not4 Immunoprecipitation and Western Blot Analysis
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Log-phase cells were harvested, washed, and stored at −80°C. Cells were disrupted by bead beating using a BeadBlaster 24 (Benchmark Scientific) in lysis buffer (0.2 M Tris base, 0.39 M ammonium sulfate, 10 mM MgSO4, 1.0 mM EDTA, 20% glycerol, 1× Protease inhibitor cocktail at pH 7.9). The clarified lysate was dialyzed in dialysis buffer (20 mM HEPES, 10 mM MgOAc, 200 mM KOAc, 2 mM EGTA, 20% glycerol, 1× Protease inhibitor cocktail at pH 7.9) for 3–4 h at 4°C. Protein (0.75 mg) was diluted to 2 mg/mL using dialysis buffer and incubated with Not4 antiserum and protein-A sepharose overnight at 4°C. Beads were washed three times with immunoprecipitation wash buffer (dialysis buffer containing 0.03% NP-40) and once with NaCl immunoprecipitation wash buffer (25 mM Tris-HCL at pH 7.5 , 0.10 M NaCl, 2 mM EDTA, 10% glycerol, 0.03% NP-40). Samples were loaded onto 7.5% SDS-PAGE gel and transferred to membranes. Blots were probed with antimyc (BioLegend, 9E10) and Not4 antiserum. One-fifteenth of the input sample was loaded to the gel.
4
Avian Influenza Virus Pathogenesis in Ducks
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Ducks aged 2 or 4 weeks old were intranasally infected with 0.25 mL of 106 EID50/mL of A/Waterfowl/Korea/S57/2016 (clade 2.3.4.4.). On day 3 p.i., the surviving ducks (n = 3 per group) were euthanized with T61 (Intervet, Canada). Lung tissues were collected in 10% suspension in PBS (pH 7.4) supplemented with 1× antibiotic antimycotic solution (Sigma-Aldrich, St. Louis, MO, USA) and homogenized with BeadBlaster 24 (Benchmark, NJ, USA). The homogenized samples were centrifuged for 3 min at 13,000 rpm, and the supernatants were used for viral titers in plaque-forming units using MDCK cells. We selected a 3-day p.i. time point to euthanize the ducks as most of the ducks that were less than 3 weeks old had already died.
5
Colorectal MPO Activity Quantification
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Snap-frozen longitudinal sections of colon were homogenized by means of a BeadBlaster 24 (Benchmark Scientific) in a 13.7 mM solution of hexadecyltrimethylammonium bromide (HTAB; Sigma Aldrich) in 50 mM phosphate buffer. Aliquots of the supernatants were tested for their ability to oxidize o-dianisidine (Sigma Aldrich) in the presence of hydrogen peroxide and compared to the activity of different MPO standards run in parallel. Resulting MPO activity was corrected to the initial weigh of each sample.
6
Evaluating Antibody Efficacy Against SARS-CoV-2 in Humanized Mice
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All work with SARS-CoV-2 was performed in a BSL3 facility. Six- to eight-week-old female BALB/c mice (Jackson Laboratory) were administered an adenovirus expressing human ACE2 (AdV-hACE2) via the intranasal route at 2.5 × 108 PFU per mouse in a final volume of 50 μl. Five days later, each respective antibody was administered via the intraperitoneal route at 10 mg/kg in a 100-μl volume. Two hours later, mice were infected with 105 PFU of SARS-CoV-2 intranasally. Mice were humanely sacrificed on day 3 and day 5 to assess the viral titers in the lungs. Lungs were homogenized using a BeadBlaster 24 (Benchmark) homogenizer. Each lung homogenate was tested in a classical plaque assay as described previously (44 (link), 47 (link)).
7
Metagenomic DNA Extraction from Fecal Samples
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Upon arrival, the samples were processed within 24 h. Where brief storage was necessary before processing, the DNA/RNA shield tubes were stored in a fridge. After processing, the remaining samples were immediately destroyed by submerging in a hypochlorite disinfectant and discarded in compliance with Human Tissue Act 2004. Genomic DNA was extracted and purified using Zymo Quick-DNA Faecal/Soil microbe kits (Zymo Research, Irvine, CA, USA), an ultra-high-density BashingBeads™ fracture resistant that omits or reduces the use of organic denaturants and proteinases [87 ]. Briefly, about 150 mg of the faecal sample was added to the BashingBead™ lysis tube and buffer. Combined chemical and mechanical lysis was then achieved by vortexing at 5 m/s, for 1 min in 5 cycles with a 30 s interval at 25 °C using Bead Blaster 24 (Benchmark Scientific, Sayreville, NJ, USA). The lysed DNA was separated from the cell debris by centrifugation (10,000× g). Thereafter, series of filtration lysis, DNA prewashing, gDNA washing, and DNA elution steps were performed. Finally, the eluted DNA was measured using a Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The DNA samples were stored at −20 °C for metagenomic sequencing downstream applications.
8
SARS-CoV-2 Infection Model in Vaccinated Mice
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All animal procedures were performed by following the Institutional Animal Care and Use Committee (IACUC) guidelines of the Icahn School of Medicine at Mount Sinai IACUC and according to an approved protocol. Six- to 8-week-old female, BALB/c mice were vaccinated via the intramuscular route with 3 μg of each respective protein with 1:1 mixture of Addavax (InvivoGen) in a total volume of 50 μL. After 3 weeks, mice were bled and vaccinated with the same protein as used for the initial vaccination. After 3 weeks, mice were administered anesthesia via the intraperitoneal route and intranasally transduced with AdV-hACE2 at 2.5 × 108 PFU per mouse (21 (link)). Anesthesia was prepared using 0.15 mg/kg of body weight ketamine and 0.03 mg/kg xylazine in water. Five days later, all mice were infected with authentic wild-type SARS-CoV-2 intranasally with 1 × 105 PFU. Mice were humanely euthanized on day 3 and day 5 for assessment of virus in the lungs. Lungs were homogenized using a BeadBlaster 24 (Benchmark) homogenizer (46 (link)– (link)48 (link)). Viral load in the lung was quantified via a classic plaque assay (49 (link), 50 (link)). For mice that also received mRNA-LNP vaccine, the vaccine was administered via the intramuscular route and each mouse was immunized with a single dose of 3 μg of mRNA-LNP.
9
Prion Disease Induction in C57Bl/6 Mice
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C57Bl/6 (Jackson Laboratory) mice were intracranially inoculated with 30µl of 1% 22L or Rocky Mountain Laboratories (RML) strains of mouse-adapted prions, or normal brain homogenate (NBH). Mice were monitored for weight loss and clinical signs of prion disease and euthanized after showing signs of terminal illness. 20% brain homogenates in phosphate-buffered saline (PBS) were made using beads and a tissue homogenizer (Benchmark Bead Blaster 24) and stored at -80C. Brain homogenates were aliquoted and treated with UV light for 30 min to sterilize before being used for cell culture.
10
Virus Titration in Lung Homogenates
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