5 mm stainless steel bead

Manufactured by Qiagen

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5 mm stainless steel beads are solid, spherical particles made of stainless steel. They are used in various laboratory applications that require mixing, milling, or homogenization of samples.

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Lab products found in correlation

149 protocols using 5 mm stainless steel bead

RNA Extraction from Mouse Tissues

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Tissues were collected from mice and placed in 600 μL of RNAlater (MilliporeSigma: #R0901500ML) and stored at −80°C. For processing, 20–30 mg of tissue was taken and placed into a 2 mL tube with 600 μL of RLT buffer with 1% β–mercaptoethanol and a 5 mm stainless steel bead (Qiagen, Hilden, Germany: #69989). Tissues were then dissociated using a Qiagen TissueLyser II (Qiagen) with the following cycle: two min dissociation at 1800 oscillations/min, one min rest, two min dissociation at 180 oscillations/min. Samples were then subject to centrifugation at 13,000 rpm for 10 min at room temperature and supernatant was transferred to a new 1.5 mL tube. RNA extractions were performed using a Qiagen RNeasy Plus Mini Kit (Qiagen: #74134), according to the manufacturer’s instructions, with an additional on-column DNase treatment (Qiagen: #79256). RNA was eluted in 30 μL of RNase/DNase free water.

Kenney D.J., O’Connell A.K., Turcinovic J., Montanaro P., Hekman R.M., Tamura T., Berneshawi A.R., Cafiero T.R., Al Abdullatif S., Blum B., Goldstein S.I., Heller B.L., Gertje H.P., Bullitt E., Trachtenberg A.J., Chavez E., Nono E.T., Morrison C., Tseng A.E., Sheikh A., Kurnick S., Grosz K., Bosmann M., Ericsson M., Huber B.R., Saeed M., Balazs A.B., Francis K.P., Klose A., Paragas N., Campbell J.D., Connor J.H., Emili A., Crossland N.A., Ploss A, & Douam F. (2022). Humanized mice reveal a macrophage-enriched gene signature defining human lung tissue protection during SARS-CoV-2 infection. Cell Reports, 39(3), 110714.

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Aedes aegypti Mosquito Infection Dynamics

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Aedes aegypti mosquitoes (Poza Rica, Mexico, P20, obtained from Dr. Gregory Ebel, Colorado State University) were infected via artificial blood meals with high or low viral loads (10⁶ or 10⁴ PFU/ml, respectively). Briefly, viruses were mixed 1:2 with pre-washed rabbit whole blood (BioIVT) supplemented with 5 mM ATP. Female mosquitoes were allowed to feed on 37 °C blood meals through an artificial membrane for 60 to 90 min. Engorged females were identified, sorted and incubated at 28 °C with 10% sucrose ad libitum for 7 day or 14 days for high and low viral loads infections, respectively. After incubations, legs and wings were removed, and placed in 2 ml round bottom tubes with 500 μl of PBS containing one 5 mm stainless steel bead (QIAGEN), homogenized with the Tissue-Lyser II (QIAGEN), and debris was pulled down at 1200 rpm for 10 min. Viral titers in sample homogenates were determined by plaque assay using Vero cells.

Noval M.G., Rodriguez-Rodriguez B.A., Rangel M.V, & Stapleford K.A. (2019). Evolution-driven attenuation of alphaviruses highlights key glycoprotein determinants regulating viral infectivity and dissemination. Cell reports, 28(2), 460-471.e5.

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Mosquito Abdominal DNA Extraction

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Mosquito abdomens were extracted individually. Samples were homogenised using a Qiagen TissueLyser II (Qiagen, Manchester, UK) with a 5 mm stainless steel bead (Qiagen) placed in each sample tube in a 96-well plate format. Once homogenised, DNA was then extracted using the Qiagen DNeasy 96 kits (Qiagen) following manufacturer’s protocol. Blood meals preserved on FTA cards were punched out using a sterile steel 4 mm radius punch. Resulting punches were incubated in ATL buffer and Proteinase K for 6 h before DNA extraction was performed following manufacturer’s protocol. Extracted DNA was stored at -20 °C until analysed.

Orsborne J., Furuya-Kanamori L., Jeffries C.L., Kristan M., Mohammed A.R., Afrane Y.A., O’Reilly K., Massad E., Drakeley C., Walker T, & Yakob L. (2019). Investigating the blood-host plasticity and dispersal of Anopheles coluzzii using a novel field-based methodology. Parasites & Vectors, 12, 143.

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Mycobacterial DNA Extraction Protocol

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25 milligrams (mg) of suspicious lesions or visible lesion containing sample material was cut out with a scalpel from the tissue or lymph nodes and suspended in 200 μl Tissue Lysis Buffer of the High Pure PCR Template Preparation kit from Roche, Rotkreutz, Switzerland. Bead beating was performed by transferring the lysis material into a 2 ml safe lock centrifugation tube (Eppendorf, Hamburg, Germany) containing a 5 mm stainless steel bead (Qiagen, Hilden, Germany). For mechanical burst of the mycobacterial cell wall, the tube was shaken 2 times for 3 min at 30 Hertz (Hz) using the Mixer Mill 301 (Retsch, Haan, Germany). The obtained suspension was used for proper DNA extraction as recommended by the manufacturer of the High Pure DNA Template Preparation kit.

Leth C., Varadharajan A., Mester P., Fischaleck M., Rossmanith P., Schmoll F, & Fink M. (2017). Matrixlysis, an improved sample preparation method for recovery of Mycobacteria from animal tissue material. PLoS ONE, 12(7), e0181157.

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Lung Tissue Homogenization Protocol

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Lungs were harvested at designated time points and washed in 1 x PBS prior to placement in 2 mL tubes containing 500 μL of sterile 1xPBS and one 5 mm stainless steel bead (QIAGEN). Lung tissue was homogenized with the Retsch MM400 mixer at 30 Hz for 2 minutes. Following homogenization, lung debris was centrifuged at 15,000 RPM for 10 minutes. Supernatants were transferred to new tubes.

Ural B.B., Yeung S.T., Damani-Yokota P., Devlin J.C., de Vries M., Vera-Licona P., Samji T., Sawai C.M., Jang G., Perez O.A., Pham Q., Maher L., Loke P., Dittmann M., Reizis B, & Khanna K.M. (2020). Identification of a nerve-associated, lung-resident interstitial macrophage subset with distinct localization and immunoregulatory properties. Science immunology, 5(45), eaax8756.

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Tissue Lysis and Protein Extraction

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All biopsies were thawed on ice. A 5 mm stainless steel bead (Cat. #69989, Qiagen, Hilden, Germany) was placed into each Eppendorf tube that was to contain a sample and these tubes were then stored at −20°C. One hundred and twenty microliters RIPA buffer (50 mM Tris Base pH 7.4, 50 mM Tris-HCL pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate) containing 1 mg/ml of protease inhibitor (Cat. #04693116001, cOmplete—Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland) was added to each tube together with a biopsy. All tubes were subsequently placed into a TissueLyser LT (Qiagen, Hilden, Germany) at 50 Hz for 5 min, centrifuged at 15,700 g for 5 min at 4°C, after which the supernatant was collected. The Pierce BCA Protein Assay Kit (Cat. #23227, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used to determine protein concentration according to kit protocol.

Lushnikova A., Bohr J., Wickbom A., Münch A., Sjöberg K., Hultgren O., Wirén A, & Hultgren Hörnquist E. (2021). Patients With Microscopic Colitis Have Altered Levels of Inhibitory and Stimulatory Biomarkers in Colon Biopsies and Sera Compared to Non-inflamed Controls. Frontiers in Medicine, 8, 727412.

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Mycobacterial DNA Extraction Protocol

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For ML qPCR the bacteria pellet resulting from ML was resuspended in 180 μl Lysis Buffer ATL of the kit DNeasy^®Blood&Tissue Kit from Qiagen, Hilden, Germany. For the homogenate qPCR, 1 ml of homogenized sample material (Fig 1) was centrifuged for 25 min at 16000 x g; the supernatant discarded and the pellet resuspended in 180 μl Lysis Buffer ATL of the DNeasy^®Blood&Tissue Kit. To perform bead beating, the lysed sample material was transferred into a 2 ml safe lock centrifugation tube (Eppendorf) containing a 5 mm stainless steel bead (Qiagen). For mechanical burst of the mycobacterial cell wall the lysis material was treated as described by direct qPCR using the Mixer Mill 301. The obtained suspension was used for proper DNA extraction as recommended by the manufacturer of the DNeasy^®Blood&Tissue kit.

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Quantification of Telomere-Related Gene Expression

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Relative mRNA levels were determined by qPCR. Total RNA was isolated from the brain tissue of the control (n = 6) and WF-exposed (n = 6) rats using a RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA), according to kit instructions. Briefly, 50–60 mg of brain tissue was homogenized in buffer RLT and one 5-mm stainless steel bead (Qiagen Inc, USA) using a tissue Lyser II (Qiagen Inc, USA). The tissue homogenate was centrifuged, and the RNA present in the supernatant was extracted and purified using RNeasy columns. RNA concentration was measured using the Nano-Drop 2000 spectrophotometer and reverse transcribed to synthesize cDNA from using random hexamers and Superscript III (Invitrogen, Carlsbad, CA, USA). cDNA was used for gene expression. Hypoxanthine-guanine phosphoribosyltranferase (HPRT) was used as the endogenous control. Gene expression was determined by standard 96-well technology using the StepOne Plus (Applied Biosystems, Carlsbad, CA) with pre-designed Assays-on-Demand TaqMan probes and primers including Trf1 and 2 (Rn01749291_m1, Rn01432601_m1), ATM (Rn01421973_m1), APP (Rn00570673_m1) (Thermo Fisher Scientific, Waltham, MA). In 96 well plates, cDNA was used for gene expression.

Shoeb M., Mustafa G.M., Kodali V.K., Smith K., Roach K.A., Boyce G., Meighan T., Roberts J.R., Erdely A, & Antonini J.M. (2019). A possible relationship between telomere length and markers of neurodegeneration in rat brain after welding fume inhalation exposure. Environmental research, 180, 108900.

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RNA Extraction and RT-qPCR Analysis

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RNA was extracted using the RNeasy Mini kit (QIAGEN, 74106). For tissue lysis, a 5 mm stainless steel bead (QIAGEN, 69989) was used to facilitate tissue lysis prior to RNA extraction. Following RNA extraction, up to 2.5 µg of RNA was used with the SuperScript Vilo cDNA synthesis kit (ThermoFisher, 11754050). cDNA was diluted to a concentration of 250 ng/µL and the RT-qPCR reactions were conducted with Thermo Scientific ABsolute Blue qPCR SYBR (ThermoFisher, AB4322B). Duplicate samples per condition were analyzed on an Applied Biosystems QuantStudio 3 qPCR instrument with all experiments being repeated 3 independent times. beta-Actin was used as reference and log fold change was calculated using the ddCT method comparing treatments to a control sample.

Upadhyayula P.S., Higgins D.M., Mela A., Banu M., Dovas A., Zandkarimi F., Patel P., Mahajan A., Humala N., Nguyen T.T., Chaudhary K.R., Liao L., Argenziano M., Sudhakar T., Sperring C.P., Shapiro B.L., Ahmed E.R., Kinslow C., Ye L.F., Siegelin M.D., Cheng S., Soni R., Bruce J.N., Stockwell B.R, & Canoll P. (2023). Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism. Nature Communications, 14, 1187.

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Total RNA Extraction from Organ Tissues

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Total RNA was extracted using the RNeasy^® Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Briefly, 100 mg of organs (if possible) were added to 1 mL QIAzol Lysis Reagent containing one 5 mm stainless steel bead (Qiagen). Tissues were disrupted and homogenized using a tissue lyser for 2 min at 20 Hz, and homogenates were incubated at room temperature (RT) for 5 min. Then, 200 µL of chloroform (Sigma, St. Louis, MO, USA) was added. After incubation for 5 min at RT, the homogenates were centrifuged at 12000g for 15 min at 4 °C. The upper aqueous phase containing RNA was transferred to a new tube, and then the QIAcube was used per the manufacturer’s protocol for animal tissue (Qiagen). Total RNA samples were stored at −80 °C until use.

Gautier C., Dufour E., Dupré C., Lizzo G., Caignard S., Riest-Fery I., Brasseur C., Legros C., Delagrange P., Nosjean O., Simonneaux V., Boutin J.A, & Guenin S.P. (2018). Hamster Melatonin Receptors: Cloning and Binding Characterization of MT1 and Attempt to Clone MT2. International Journal of Molecular Sciences, 19(7), 1957.

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