Bead mill homogenizer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Bead Mill Homogenizer is a lab equipment used for efficient disruption and homogenization of biological samples. It utilizes high-speed agitation of samples with beads to effectively break down cells, tissues, and other solid materials.

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11 protocols using bead mill homogenizer

Insulin Signaling Pathway Analysis

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Two to three months old male mice were fasted overnight. Following anesthesia, mice were injected with 6 nmol Humulin (Eli Lilly) or 9 nmol S597 analogs per mouse via inferior vena cava. Livers were removed 3 min after injection. White adipose tissue and skeletal muscles were removed at 5 and 7 min after injection, respectively. Tissues were mixed with the lysis buffer B supplemented with cOmplete Protease Inhibitor Cocktail (Roche), PhosSTOP (Sigma), and 25 U/ml turbo nuclease (Accelagen), homogenized with Fisherbrand^TM Bead Mill homogenizer, and then incubated on ice for 1 h. After centrifuging at 20,817×g at 4 °C for 30 min, the concentrations of cell lysate were measured using Micro BCA Protein Assay Kit (Thermo Scientific). The lysates were then analyzed by quantitative western blotting.
For IR endocytosis assays, the livers were fixed in 10% Neutral Buffered Formalin (NBF) and embedded in paraffin blocks. Sections were deparaffinized and subjected to immunohistochemistry as described in the immunohistochemistry section and previous study^{48 (link)}.

Park J., Li J., Mayer J.P., Ball K.A., Wu J., Hall C., Accili D., Stowell M.H., Bai X.C, & Choi E. (2022). Activation of the insulin receptor by an insulin mimetic peptide. Nature Communications, 13, 5594.

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Gene Expression Analysis of Macrophage Phenotypes

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Approximately 30–60 mg of tissue was homogenized in 1 mL Trizol using a Bead Mill Homogenizer (Fischer Scientific, Hampton, NH). Extracted RNA was precipitated overnight at −20 °C, and subsequently pelleted, dried, and re-suspended in 20 μl of sterile water, as previously described^{50 (link)}. Total RNA (1 µg) was reversed transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, MA). Quantitative PCR was performed using the BioRad CFX386 QPCR System (Hercules, CA) with Taqman Universal PCR Master Mix (Applied Biosystems). β2 microglobulin (β2M) was used as the housekeeping gene, and all data are represented relative to its expression using the ΔΔCq method^{51 (link)}. CON + SED was used as a reference group for relative gene expression. Taqman primers for β2M, Emr-1 (F4/80), macrophage mannose receptor 1 (MRC-1/CD206), tumor necrosis factor α (TNFα), interleukin 6 (IL-6), transforming growth factor β1 (TGFβ1), and collagen 1α (Col1α) were all purchased from Applied Biosystems. Gene expression assay ID numbers are provided in Supplementary Table 1.

D’Souza D., Roubos S., Larkin J., Lloyd J., Emmons R., Chen H, & De Lisio M. (2019). The Late Effects of Radiation Therapy on Skeletal Muscle Morphology and Progenitor Cell Content are Influenced by Diet-Induced Obesity and Exercise Training in Male Mice. Scientific Reports, 9, 6691.

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Tissue Sampling and Blood Analysis

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Tissues were collected between 9:00 and 11:00 on day 6 from all mice. Mice were anesthetized with an intraperitoneal injection of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine, and they were then exsanguinated via blood collection from the inferior vena cava. For complete blood count (CBC), blood was collected into Sarstedt EDTA microtubes and tested on an Advia 120 analyzer (Siemens Healthineers, Forchheim, Bavaria, Germany). Serum samples were collected by centrifuging whole blood in Sarsedt Z-Gel microtubes at 17,000×g for 15 min.
Whole brains were extracted and flash frozen in liquid nitrogen immediately after exsanguination and decapitation. Brains were homogenized using a bead-mill homogenizer (Thermo Fischer Scientific, Waltheim, Massachusetts, USA) in ice-cold RIPA buffer (#89901, Thermo Fischer) with freshly added protease inhibitor cocktail (Millipore Sigma, Burlington, Massachusetts, USA) and centrifuged at 14,000×g for 15 min at 4 °C. Protein concentrations of lysate supernatants were determined using a Pierce BCA kit (#23227, Thermo Fischer Scientific).

Wolff B.S., Alshawi S.A., Feng L.R., Juneau P.L, & Saligan L.N. (2021). Inflammation plays a causal role in fatigue-like behavior induced by pelvic irradiation in mice. Brain, Behavior, & Immunity - Health, 15, 100264.

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Quantitative PCR Analysis of Bioprinted Chondrocytes

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Bioprinted Alg-PBA-Ca hydrogel and alginate hydrogel encapsulated with mouse chondrocytes were first depolymerized by a depolymerizing solution (Cell Applications Inc., San Diego, CA, USA) to remove Ca²⁺ crosslinking; this was followed by homogenization with a bead mill homogenizer (Fisher Scientific, Waltham, MA, USA) in the cell lysis buffer (Qiagen, Hilden, Germany) to release the cytoplasm. The total RNA was extracted from the cell lysate with the RNeasy mini-kits (Qiagen) following the protocol. The extracted total RNA was transcribed into complementary DNA (cDNA) with an iScript cDNA synthesis kit (Quantabio, Berverly, MA, USA). Real-time PCR analysis was conducted in a StepOnePlus™ Real-Time PCR System (Thermo Scientific, Waltham, MA, USA) with SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) dye. The cDNA samples were evaluated for the target genes and the housekeeping gene (Actb). The primer sequences are shown in Table S1. The relative expression of each studied gene was determined by the comparative Ct (2 ^−ΔΔCt) method [45 (link)]. Each group had three replicates.

Zhang W., Kuss M., Yan Y, & Shi W. (2023). Dynamic Alginate Hydrogel as an Antioxidative Bioink for Bioprinting. Gels, 9(4), 312.

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Hamster SARS-CoV-2 Infection Modeling Protocol

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PBS was purchased from Merck. Male Syrian golden hamsters were purchased from Janvier Labs. Swabs (1 mL Liquid Amies Regular Flocked) were purchased from Appleton Woods. GoTaq^® Probe 1-Step RT–qPCR System was purchased from Promega. SARS-CoV-2 (2019-nCoV) CDC qPCR Probe Assay, CDC RUO 2019-nCoV_N_Positive Control and the SARS-CoV-2 E SgRNA were purchased from IDT. TRIzol reagent, GlycoBlue^™, Phasemaker^™ tubes, Nanodrop and TURBO DNA-free^™ kit were purchased from Thermo Fisher. A bead mill homogenizer was purchased from Fisher Scientific. Precellys CKMix lysing tubes were purchased from Bertin Instruments. A Chromo4^™ Real-Time PCR Detector was purchased from Bio-Rad. Transmission cages were purchased from Tecniplast UK Ltd. GS-441524 was purchased from Carbosynth (UK).

Box H.J., Sharp J., Pennington S.H., Kijak E., Tatham L., Caygill C.H., Lopeman R.C., Jeffreys L.N., Herriott J., Neary M., Valentijn A., Pertinez H., Curley P., Arshad U., Rajoli R.K., Jochmans D., Vangeel L., Neyts J., Chatelain E., Escudié F., Scandale I., Rannard S., Stewart J.P., Biagini G.A, & Owen A. (2023). Lack of antiviral activity of probenecid in vitro and in Syrian golden hamsters. Journal of Antimicrobial Chemotherapy, 79(1), 172-178.

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Zebrafish Xenograft for GBM Drug Testing

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tdTomato/luciferase stably expressing GBM18 and H4 cells, generated from U18/H4 lines with a lentivirus-based approach (#32904, Addgene), were injected into AB or fli:EGFP zebrafish embryos as described.^{17 (link)} Embryos were imaged using ImageXpress Nano (Molecular Devices) and lysed or processed for cryosectioning.^{17 (link)} Cryosections were stained per routine protocols (Supplementary Table S1).  Images were taken with NikonTi2. For Blood-brain Barrier (BBB) analysis, adult zebrafish were treated for 4h and sacrificed by hypothermic shock. Following decapitation, brains were removed and snap-frozen. Brain tissue was homogenized using a bead-mill homogenizer (Fisherbrand) and subjected to LC/MS-MS. Detected drug concentrations were normalized against water-tank drug concentrations.

Zisi A., Kanellis D.C., Moussaud S., Karlsson I., Carén H., Bräutigam L., Bartek J, & Lindström M.S. (2022). Small molecule-mediated disruption of ribosome biogenesis synergizes with FGFR inhibitors to suppress glioma cell growth. Neuro-Oncology, 25(6), 1058-1072.

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Metabolite Extraction from Tumors

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Tumors were weighed and metabolites extracted in 1 ml 80% MeOH (−80 °C) using a Fisherbrand™ Bead Mill Homogenizer. Samples were spun twice at >17,000 g (4 °C) to remove debris. Extraction volumes equivalent to 3 mg of tissue were normalized to 500 ul with 80% MeOH and evaporated using a Genevac EZ-2 evaporator. Evaporated samples were stored at −80 °C.

Krall A.S., Mullen P.J., Surjono F., Momcilovic M., Schmid E.W., Halbrook C.J., Thambundit A., Mittelman S.D., Lyssiotis C.A., Shackelford D.B., Knott S.R, & Christofk H.R. (2021). Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth. Cell metabolism, 33(5), 1013-1026.e6.

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Cell Lysis and Protein Extraction

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Proteins were extracted in 1 ml cell lysis buffer (see above) using a Fisherbrand™ Bead Mill Homogenizer. Samples were spun twice at >17,000 g (4 °C) to remove debris, and stored at −80 °C.

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Lipid Extraction from Serum and Tissue Samples

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The lipid extraction procedure of serum samples was based on Bligh and Dyer (1959) (link). In Brief, 100 μL of serum sample was extracted with 800 μL of cold chloroform/methanol 1:1 (v/v, with IS) twice, followed by drying under vacuum. For tissues, lipids were extracted by the modified method of Folch (1957) . Briefly, approximately 10 mg of tissue sample was weighed followed by adding 600 μL of ice-cold chloroform/methanol 2:1 (v/v, with IS) and homogenized using a Bead Mill Homogenizer (Fisherbrand®, Pittsburgh, PA). The extraction was performed twice for each sample, and the combined extract was dried under vacuum. Then, the dried lipids were dissolved in 100 μL of methanol, centrifuged at 2500×g 4 °C for 15 min to remove any insoluble material, and thereafter stored at -80 °C until analysis. The sample pretreatment was completed within 1 h to avoid lipid auto-oxidation or degradation.

Chen Z., Liang Q., Wu Y., Gao Z., Kobayashi S., Patel J., Li C., Cai F., Zhang Y., Liang C., Chiba H, & Hui S.P. (2020). Comprehensive lipidomic profiling in serum and multiple tissues from a mouse model of diabetes. Metabolomics : Official journal of the Metabolomic Society, 16(11).

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Bacterial Burden Quantification

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After the animals were euthanized and enucleated, the whole eyes were homogenized and CFU were enumerated to determine the bacterial burden (CFU/mL). To do so, samples were suspended in tubes containing 1.4 mm ceramic beads (Fisher Scientific, Hampton, NH) and 0.5 ml phosphate-buffered saline (PBS) and homogenized using the Fisher brand Bead Mill homogenizer. Aliquots (0.1 ml) were then removed, serially diluted in 0.8% sodium chloride, and plated on mannitol salt agar plates (Fisher Scientific, Hampton, NH) for enumeration after incubating at 37°C for 16 hours.

Meijome T.E., Wozniak R., Kang L., Azzouz L., Niziol L.M., Johnson W.L., Kriegel M, & Woodward M.A. (2022). Assessment of Keratitis Severity Using Quantitative Image Analysis in an In Vivo Murine Model of Staphylococcus aureus Bacterial Keratitis. Translational Vision Science & Technology, 11(11), 12.

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