A Calpain Activity Assay Kit (Abcam, Cat# ab65308) was used to quantify relative calpain enzyme activity. At the end of the ex vivo exposure, sciatic nerves were transferred to BioMasher tubes (Takara Bio USA), snap frozen in liquid nitrogen, and stored at −80°C. Immediately prior to performing the calpain activity assay, sciatic nerves were pulverized using a pestle (BioMasher, Takara Bio USA) under liquid nitrogen and then ice-cold extraction buffer was added. Samples were centrifuged at 4°C at 21,000 × g for 5 min. Supernatants were transferred to a fresh, ice-cold tube, and protein content was quantified using a Pierce BCA Protein Assay (Thermo Scientific, Cat# 23225). Calpain activity was measured in 25 to 50 µg of total protein for each sample according to manufacturer protocol. Changes in relative fluorescent units were detected using a Synergy H1 microplate reader (BioTek Instruments). Relative fluorescence units for each sample were blank-subtracted, divided by the total protein loaded, and normalized to the average of vehicle controls within each experiment. Calpain activity is reported as the percentage of vehicle control.
Biomasher
Manufactured by Takara Bio
Sourced in Japan, United States
The BioMasher is a laboratory equipment designed for efficient sample preparation. It is used to homogenize and disrupt biological samples, such as tissues or cells, to extract their contents for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Direct zol rna miniprep kit, by Zymo Research (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Db ffap 123 3253, by Agilent Technologies (2 mentions)
Propionic acid, by Merck Group (2 mentions)
Butyric acid, by Merck Group (2 mentions)
Acetic acid, by Merck Group (2 mentions)
Mm01228299 m1, by Thermo Fisher Scientific (2 mentions)
Qiashredder, by Qiagen (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Synergy h1 microplate reader, by Agilent Technologies (1 mentions)
Calpain activity assay kit, by Abcam (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Flame ionization detector, by Hewlett-Packard (1 mentions)
0.45 μm syringe filter, by Advantec (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
12 protocols using biomasher
1
Quantifying Calpain Activity in Sciatic Nerves
2
qRT-PCR Analysis of ΔNp63 Knockout
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Total RNA was extracted from ΔNp63fl/fl (control) and Acta2CreERT2;ΔNp63fl/fl knockout (ΔNp63MECcKO) mouse submandibular glands 6 months following TAM administration were homogenized in Trizol reagent (Invitrogen) using BioMashers (TaKaRa). The RNA was phase separated by chloroform and further isolated using the Direct-zol RNA Miniprep kit (Zymo Research). cDNA was synthesized by reverse transcribing isolated RNA using the iScript cDNA Synthesis kit (Bio-Rad). qRT-PCR (quantitative Real-Time Reverse Transcription- Polymerase Chain Reaction) was performed as previously described [21 (link)]. Briefly, qRT-PCR was performed on a CFX96 Touch Real-Time PCR machine using iQ SYBR Green Supermix (Bio-Rad) in triplicates in at least three independent biological replicates. Relative expression values of each target gene were normalized to hypoxanthine guanine phosphoribosyltransferase (Hprt) expression. Primer sequences are shown in Supplementary Table 1 . n = 3 for each sex.
3
Quantitative Real-Time PCR Protocol
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Total RNA was extracted by resuspending the mSGc in Trizol reagent (Thermo Fisher Scientific) using BioMashers (TaKaRa). The RNA was phase separated by chloroform and further isolated using the Direct-zol RNA Miniprep kit (Zymo Research). Isolated RNA was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer’s instructions and qRT-PCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad). All qRT-PCR assays were performed in triplicates in at least three independent experiments. Relative expression values of each target gene were normalized to hypoxanthine guanine phosphoribosyltransferase (Hprt) expression. See S1 Table for primers sequences.
4
Quantifying Transgenic Protein Expression
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After sacrificing mice which received systemic administration of AAV four weeks prior, brains were dissected and split into individual hemispheres while peripheral organs were harvested and immediately frozen at -80 ℃ for transgenic protein quantification by enzyme-linked immunosorbent assay (ELISA). Tissues were thawed on ice before homogenization using either disposable micro tissue homogenizers for brain hemispheres (BioMasher, Takara Bio USA, Inc., San Jose, CA, USA) or a motorized tissue homogenizer with disposable pestles for peripheral organs. Tissues were homogenized in a volume of sterile 1x PBS normalized to the weight of tissue at a ratio of 1 g of tissue to 10 mL of PBS. After homogenization, samples were immediately processed by ELISA (GFP ELISA Kit, Fluorescent, #ab229403, Abcam Inc., Waltham, MA, USA) according to the manufacturer's instructions. Reported GFP concentrations represent picograms per milliliter of tissue homogenate.
5
Fungal Detection in Nasal Polyps
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After the nasal polyps were disrupted with glass beads and 100 μL of Tris-EDTA (10 mM Tris-Cl, 1 mM ethylenediaminetetraacetic acid pH 8.0) using a homogenizer (BioMasher) (Takara Bio Inc., Otsu, Japan), and 1 μL of homogenized polyp samples were spread onto 5% sheep blood agar, chocolate agar, Columbia anaerobic blood agar, Drigalski agar, and Sabouraud agar, and incubated for 2 weeks at 35°C. All specimens were stained with Grocott methanamine silver staining for confirmation of the existence of fungal organisms.
6
Quantifying Short-Chain Fatty Acids in Mouse Cecum
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To evaluate the SCFA content of the mouse caecum, it was ground to a concentration of 1 g/mL in an 80% methanol solution using a BioMasher (TaKaRa, Shiga, Japan). Centrifugation was performed at 13,000 rpm for 10 min at 4 °C, and the supernatant was filtered using 0.45 μm syringe filters from ADVANTEC. The analysis was performed using a flame ionization detector (Hewlett Packard, Palo Alto, CA, USA) and a GC column (DB-FFAP 123-3253, 50 mm × 0.32 mm × 0.50 μm, Agilent Technologies, Inc., Santa Clara, CA, USA). SCFA concentrations were quantified using Acetic acid, propionic acid, and butyric acid as standards. The SCFA content in the cecum was determined using a calibration curve based on the corresponding standards. Acetic acid, propionic acid, and butyric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Quantification of Cecal SCFA Levels
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To evaluate the SCFA content in the mouse cecum, we ground the tissue using the BioMasher (TaKaRa, Shiga, Japan) in an 80% methanol solution. The ground solution was then centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatant was filtered using ADVANTEC’s 0.45 μm syringe filter. Subsequently, analysis was performed using a flame ionization detector (HP-5890/5971, Hewlett Packard, Palo Alto, CA, USA) and GC column (DB-FFAP 123-3253, Agilent Technologies, Inc., Santa Clara, CA, USA, 50 mm × 0.32 mm × 0.50 μm). The SCFA levels were quantified using Acetic acid, propionic acid, and butyric acid as standards. The content of SCFAs in the cecum was determined using a calibration curve based on the respective standards. Acetic acid, propionic acid, and butyric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).
8
Extraction of Biomolecules from Mouse Feces
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Fresh mouse feces were dissolved in PBS (1 g feces/ml) at room temperature using BioMasher (TaKaRa), and filtered with a 70-mm cell strainer. The filtered extract was further filtered with a 0.45-mm syringe filter to remove bacterial and fungal bodies (fecal extract). To isolate total DNA, RNA, and protein from the fecal extract, a NucleoSpin TriPrep kit (Macherey-nagel 740966.10) was used according to the manufacturer's protocol. The A260/A280 of total RNA and DNA was 2.0 and 1.8, respectively. The protein concentration was measured with a BCA protein assay kit (TaKaRa). DNA, RNA, and protein (10 mg/ml each) were used as the ''extracted DNA,'' ''extracted RNA,'' and ''extracted protein,'' respectively.
9
Intestinal Defensins, Lysozyme, and Claudin Expression
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Mouse small intestines tissues were extracted using BioMasher (TaKaRa, Shiga, Japan) and filtrated using a QIAshredder (Qiagen, Hilden, Germany). Total RNA from the intestinal tissues was isolated and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany), followed by cDNA synthesis using the RevertAid First-Strand cDNA synthesis kit (Fermentas, MA, USA). The qRT-PCR quantification was performed using Mm02524428_g1(α-defensin-1), Mm01228299_m1 (lysozyme), and Mm01342184_m1 (claudin1) TaqMan primer sets (Applied Biosystems, Foster City, CA, USA). The amplification conditions were determined using the Quant 3 PCR system (Applied Biosystems).
10
Rat Mesenteric Artery Transcriptome
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The tissues of mesenteric arteries were harvested from each rat body under sodium pentobarbital anaesthesia [50 mg/kg intraperitoneal (i.p.)] at 6 weeks of age. Fat and connective tissues were quickly removed from the samples using forceps and scissors, and the isolated arteries were stored in RNAlater stabilization solution (Qiagen GmbH, Hilden, Germany) at −20°C until use. Two days after harvesting, two to three pieces of arterial tissue were cut to 2–3 mm in size, homogenized using a BioMasher (Takara Bio Inc., Shiga Prefecture, Japan) with 5 mm diameter glass beads and a Qiagen TissueLyser (Qiagen GmbH). Total RNA was extracted with a miRNeasy Mini kit (Qiagen) according to the manufacturer's protocol. RNA quality was evaluated using a RNA Nano Chip and Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbornn, Germany), and RNA of sufficient quality was used for microarray experiments. Tissue samples taken from four of the eight rats per group were used in the microarray analyses.
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