4 to 6 week-old Xbp1flox/floxErn1flox/flox ESR Cre+ and Cre− littermate mice were given 5 consecutive daily doses of 75 mg/kg tamoxifen (MilliporeSigma) in corn oil intraperitoneally (i.p.) to induce expression of Cre recombinase. After resting for 3 days, mice were infected i.p. with 1E4 PFU of mouse adapted ZIKV Dakar [28 (link)]. Animals received 1.5 mg interferon receptor blocking monoclonal antibody MAR1-5A3 (Leinco, catalog number I-401) i.p. the day prior to infection and 1 mg the day after infection. Mice were euthanized 3 days after infection. Harvested tissues were immediately frozen on dry ice and stored at −80 °C until processing. Samples were homogenized in TRI Reagent (Zymo Research) with Lysing Matrix D beads (MP Biomedicals) on a FastPrep tissue homogenizer (MP Biomedicals). All procedures performed in this study were approved by the University of Washington Institutional Animal Care and Use Committee (28 December 2016).
Fastprep tissue homogenizer
Manufactured by MP Biomedicals
Sourced in United States
The FastPrep tissue homogenizer is a laboratory instrument designed for the efficient and rapid disruption of biological samples, such as tissues, cells, and microorganisms. It utilizes high-speed agitation to homogenize the sample, effectively breaking down the cellular structure and releasing the contents for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Rnalater, by Merck Group (2 mentions)
Corbett rotorgene 6000 system, by Qiagen (2 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Tamoxifen, by Merck Group (1 mentions)
Lysing matrix d beads, by MP Biomedicals (1 mentions)
Tri reagent, by Zymo Research (1 mentions)
Luminex 200 instrument, by Bio-Rad (1 mentions)
Halt protease inhibitor mixture, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Prism version 7, by GraphPad (1 mentions)
Optima l 80 xp, by Beckman Coulter (1 mentions)
Cytation 3 cell imaging reader, by Agilent Technologies (1 mentions)
Protease inhibitor tablet, by Thermo Fisher Scientific (1 mentions)
Lysing matrix d tube, by MP Biomedicals (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
13 protocols using fastprep tissue homogenizer
1
Acute ZIKV Infection Model in Mice
2
Cytokine Profiling in Myocardial Infarction
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Procarta cytokine profiling kit was used to detect 6 different rat cytokines per reaction for protein extracted from myocardial infarct area. This assay uses xMAP technology to enable the detection and quantification of multiple protein targets simultaneously [12 (link)]. Firstly, multiple samples from myocardial infarct area were homogenized simultaneously using a FastPrep tissue homogenizer (MP Biomedicals, CA, USA) for cytokines assay. Then, 50 μl/well antibody beads were added onto the Filter plate, washed by Wash buffer. Then, 50 μl/well of each sample was added, incubated for at least 1 h at room temperature and washed with Wash buffer. Afterwards, 25 μl/well of the Detection Antibody was added and the Filter plate was shaken at 500 rpm for 30 min at room temperature. After adding Sreptavidin-PE, the detection was conducted using a Luminex 200 instrument (Bio-Rad, CA, USA). In the present study, investigated cytokines were tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), intercellular adhesion monocyte chemoattractant protein-3 (MCP-3), regulated on activation normal T cell expressed and secreted (RANTES), intercellular adhesion molecule (ICAM), soluble vascular cell adhesion molecule-1 (sVCAM-1).
3
Multiplex Cytokine Analysis of Tumor Lysates
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For the cytokine analysis, a portion of each tumor was lysed and homogenized with radioimmunoprecipitation assay lysis and extraction buffer (Thermo Fisher) containing Halt protease inhibitor mixture (Thermo Fisher) in a Fast Prep tissue homogenizer (MPBio). After lysis and homogenization, protein concentration was determined by using a bicinchoninic acid assay kit (Thermo Fisher). Each lysate (1 μg/well) was analyzed for murine MCP-1, MIP-1α, and MIP-1β by MSD multiplex assay.
4
Extracting DNA from Museum Skulls
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Because DNA methylation is highly tissue-specific, it is necessary to standardize the tissue sampled across individuals. We sampled bone tissue from traditionally prepared dried museum skulls. To minimize damage to the skulls, we sampled microturbinates (small nasal bones) by inserting a sterile micropick into the nasal cavity to dislodge 5–12 mg of tissue (Wisely et al. 2004 ; Taylor and Hoffman 2010 ). Prior to DNA extraction, the bone fragments were placed into thick-walled 2 mL microcentrifuge tubes with four 2.4 mm stainless steel beads and processed in a FastPrep tissue homogenizer (MP Biomedicals) for 1 min at 6.0 m/s. DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit, with modifications for working with museum specimens (see Rubi et al. 2020 (link) for full extraction protocol). All pre-amplification steps were performed under a laminar flow hood in the Ancient DNA Laboratory at the University of Michigan, a dedicated laboratory for processing low quality specimens, and followed stringent anti-contamination protocols (unidirectional flow of equipment and personnel, filtered pipette tips, additional negative controls, etc.).
5
Quantifying Gene Expression Using RT-qPCR
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For RT-qPCR analysis, SMGs stored in RNAlater® (R0901-100ml, Sigma-Aldrich) were homogenized using a FastPrep™ tissue homogenizer (MP Biomedicals Santa Ana, CA) and RNeasy® Micro Kit (74004, Qiagen) was used for total RNA extraction. RNA concentration as well as the A260/280 and A260/230 ratios were then measured with the NanoDrop ND-1000 Spectrophotometer (Thermo Fischer Scientific, Nottingham UK). iScript™ cDNA Synthesis kit (170-8890, Bio-Rad) was used to reverse transcribe 100 ng of extracted RNA. PCR reactions (10 µl/well) were prepared by adding SsoAdvanced ™ Universal SYBR Green Supermix (172–5271, Bio-Rad), primers (PrimerDesign™, Ltd. mouse AURKB, K5, c-Kit, AQP5, NKCC1, ANO1, β-Actin or GAPDH) and cDNA template. Thermal cycling was performed using Corbett RotorGene 6000 System (Qiagen, UK). In all RT-qPCR experiments, relative gene quantification was assessed according to the following equation: ΔΔCT = [Ct GOI Exp − Ct HKG Exp] − [Ct GOI Cal − Ct HKG Cal]: Ct: cycle threshold, GOI: gene of interest, Exp: poly (I:C)-injected glands, HKG: housekeeping gene, Cal: control glands injected by the vehicle. There were 3–4 biological replicates for each experiment and the values were analysed by GraphPad Prism Version 7 (GraphPad software, USA).
6
Yeast Cell Lysis for Protein Extraction
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Cell cultures in YPD ±pheromone were spun down at 600g for 2 minutes. Cell pellets were resuspended in 400μl of ice-cold lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 2.5mM EDTA, 1% (v/v) Triton X-100, 2mM PMSF, 20mM NEM and 1 tablet of protease inhibitor cocktail per 10ml of buffer) in screw cap tubes. Acid washed glass beads (filling a 200μl pcr tube) were added to each tube abd cells were lysed using a FastPrep tissue homogenizer (MP Biomedicals) for 30s followed by 1 min on ice. Homogenization was repeated 5 times in total. Tubes were kept on ice for 20min and centrifuged at 300g for 2min at 4°C. Supernatants were transferred into fresh tubes (±400μl) and spun at 25,000 rpm for 30min at 4°C (Beckman Optima L-80XP). The supernatant was collected and the pellet was resuspended in 200μl of lysis buffer. Laemli buffer was added to the samples which were heated at 95°C for 5min.
7
Comprehensive Microbial DNA Extraction
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Total microbial DNA was extracted from biopsies in two batches using the DNeasy blood and tissue kit (Qiagen), with an additional bead beating step to ensure adequate cell lysis. Bead beating was performed using both 5 mm stainless steel beads to disrupt tissue (Qiagen 69989) and glass beads (Mo-Bio, Mississauga, ON, Canada) to disrupt bacterial cells, in conjunction with the FastPrep tissue homogenizer (MP Biomedicals, Santa Ana, CA, USA) set to speed 6 for 30 s. Additional enzymatic lysis was conducted through the addition of proteinase K (as per the Qiagen protocol) and incubation of samples at 95°C.
8
Biodistribution of Fluorescent Polymers
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Mice bearing day 18 C1498 were injected IV with fluorescently–labeled polymer variants at a dose of 80 nmol fluorophore. After 8 hours, mice were euthanized and organs were weighed and homogenized using FastPrep tissue homogenizer (MP Bio). Supernatant fluorescence intensity was quantified using Cytation3 Cell Imaging Reader (BioTek). Liver samples consist of the average of 2 ∼100 mg tissue samples from the left lobe, kidney samples are 1 whole kidney, spleen samples are the entire spleen, and plasma samples are ∼50 μL, normalized to actual volume.
9
In Vivo LDL Binding Assay in Hyperlipidemic Mice
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ApoE−/− or LDLr−/− mice that had been on a HFD for 10 weeks received 10 μg (apoE−/−) or 25 μg (LDLr−/−) of IL-10 or an equimolar equivalent of Fab-IL-10 i.v. After 2 h, 100 μl of blood was collected for in vivo LDL binding. The heart, aorta, liver, spleen, lung, and kidney were then harvested from mice following perfusion with PBS and euthanasia. Organs were homogenized using a FastPrep tissue homogenizer (MP Bio) in Lysing Matrix D tubes (MP Bio) containing Tissue Protein Extraction buffer (T-PER, Thermo Scientific) supplemented with protease inhibitor tablets (Roche). IL-10 was quantified by ELISA (Invitrogen) and normalized by total protein content from a Pierce BCA Protein Assay (Thermo Scientific).
10
Protein Extraction and Inactivation Assessment
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The cell pellet was suspended in 500 μl of the corresponding lysis buffer. In the case of SDS and SDC, samples were additionally heated for 10 min at 95 °C. An additional BB step was optionally included to test the influence of mechanical cell disruption on inactivation in the inactivation efficiency experiment. BB was performed with 0.1 mm silica beads in a FastPrep Tissue Homogenizer (MP Biomedicals, only half of the beads were used) by applying three cycles at 6.5 m/s speed for 45 s with a 90 s break between each cycle. Samples were centrifuged for 5 min at 10,000 rcf, and the supernatant was collected. Of note, only samples with BB were further processed for LC-MS/MS measurements.
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