5 mm steel bead

Manufactured by Qiagen

Sourced in Germany, United Kingdom

The 5-mm steel bead is a basic laboratory instrument used for various sample preparation processes. It serves as a physical tool to aid in the disruption, homogenization, or mixing of samples during procedures such as cell lysis, tissue dissociation, or sample homogenization. The bead is made of durable steel material and measures 5 millimeters in diameter.

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5 mm stainless steel beads (149 citations) Steel beads (12 citations) Sterile 5 mm steel bead (2 citations) TissueLyser (II—5 mm stainless steel bead, (2 citations)

25 protocols using 5 mm steel bead

Tissue Sampling and RNA Extraction for Wheat NILs

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The LD222 NILs were grown in 1 L pots in the glasshouse under long day conditions (16 h light: 8 h dark). We harvested flag leaf, as well as glume, lemma, palea, and anthers at Waddington stage 7.5–8 (Waddington et al. 1983 (link)) from central spikelets of the main spike of four plants for each NIL (four biological replicates). We also collected grains from florets one and two at 3, 10, and 20 dpa from the four central spikelets (eight grains per sample) of the main spike of four plants for each NIL (four biological replicates). All tissues were immediately frozen in liquid nitrogen and stored at − 80 °C.
Grains were homogenized using mortar and pestle with liquid nitrogen, while other tissues were homogenized in SPEX CertiPrep 2010–230 Geno/Grinder (Cat No.: 12605297, Fischer Scientific) using 5-mm steel beads (Cat No.: 69989, Qiagen). For grain samples, RNA was extracted following the protocol described in Adamski et al. (2021 (link)). For non-grain tissues, we used the Spectrum Plant Total RNA kit (Cat No.: STRN250-1KT, Sigma) following the manufacturer’s protocol.

Chen Y., Liu Y., Zhang J., Torrance A., Watanabe N., Adamski N.M, & Uauy C. (2022). The Triticum ispahanicum elongated glume locus P2 maps to chromosome 6A and is associated with the ectopic expression of SVP-A1. TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik, 135(7), 2313-2331.

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Glutathione Quantification in Lung Tissue

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Approximately 20–40 mg of lung tissue was homogenized in 400 μL of TES/SB buffer (20 mM Tris, pH 7.4, 1 mM EDTA, 250 mM sucrose, 20 mM serine, and 1 mM boric acid with 10 μL/mL protease inhibitor (Roche, Indianapolis, IN)) using 5 mm steel beads (Qiagen, Germantown, MD) in a 2 mL micro-tube for 5 min at 50 Hz using a TissueLyser (Qiagen). Levels of glutathione were measured as previously described (Weldy et al. 2011 (link)). Briefly, glutathione in lung tissue samples and standards (0–0.25 mM) was reduced with tris(2-carboxyethyl) phosphine, and then derivatized with napthelene-2,3-dicarboxaldehyde. Relative fluorescence intensity was measured for samples and standards. Sample concentrations were interpolated from the GSH standard curve. Standards and samples were analyzed in triplicate and samples were normalized to total protein levels in the lung tissue sample measured using the Bradford method.

Scoville D.K., Nolin J.D., Ogden H.L., An D., Afsharinejad Z., Johnson B.W., Bammler T.K., Gao X., Frevert C.W., Altemeier W.A., Hallstrand T.S, & Kavanagh T.J. (2019). Quantum Dots and Mouse Strain Influence House Dust Mite-Induced Allergic Airway Disease. Toxicology and applied pharmacology, 368, 55-62.

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Lymph Node Tissue Lysis and Protein Quantification

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Lymph nodes were collected at specific time points and snap-frozen in a mixture of isopropanol and dry ice and stored at −80°C until use. Frozen tissues were cut on dry ice, weighed (∼20 mg), and disrupted using the TissueLyzer® II system (QIAGEN). Tissues were disrupted using 5 mm steel beads (QIAGEN) under the following conditions: 2 × 30 Hz, 20 s per cycle. Homogenized tissues were resuspended in 750 μl of M-Per lysis buffer, in the presence of CoMplete protease inhibitor cocktail and incubated on ice for 30 minutes with inversions every 10 minutes. For the IL-6 ELISA, whole lymph nodes were resuspended in lysis buffer (as above) prior to homogenization (as above). Lysates were cleared by centrifugation (2270 g, 30 minutes, 4°C), transferred to new tubes, and stored at −80°C until use. An aliquot was diluted 1:100 in PBS and total protein content of the lysate sample was determined using the Pierce microBCA protein assay kit or measured undiluted using the Pierce BCA protein assay kit for the IL-6 ELISA.

Alameh M.G., Tombácz I., Bettini E., Lederer K., Ndeupen S., Sittplangkoon C., Wilmore J.R., Gaudette B.T., Soliman O.Y., Pine M., Hicks P., Manzoni T.B., Knox J.J., Johnson J.L., Laczkó D., Muramatsu H., Davis B., Meng W., Rosenfeld A.M., Strohmeier S., Lin P.J., Mui B.L., Tam Y.K., Karikó K., Jacquet A., Krammer F., Bates P., Cancro M.P., Weissman D., Luning Prak E.T., Allman D., Igyártó B.Z., Locci M, & Pardi N. (2021). Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses. Immunity, 54(12), 2877-2892.e7.

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Spleen RNA Extraction and cDNA Synthesis

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Total RNA from the spleen was extracted for eight fish from each tank. Tissue from each sample was homogenized using the TissueLyzer mechanical homogenizer (Qiagen, Hilden, Germany) with 5 mm steel beads (Qiagen) and the TRIzol Reagent solution (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. RNA quantity and quality were assessed by spectrophotometry (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA). Only RNA samples of high quality were used for constructing the cDNA libraries. Total RNA was used as a template to synthesize cDNA using the PrimeScript RT Reagent Kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. Approximately 1 μg RNA was used as input material.

Tzortzatos O.P., Toubanaki D.K., Kolygas M.N., Kotzamanis Y., Roussos E., Bakopoulos V., Chatzopoulos A., Athanassopoulou F, & Karagouni E. (2024). Dietary Artemisia arborescens Supplementation Effects on Growth, Oxidative Status, and Immunity of Gilthead Seabream (Sparus aurata L.). Animals : an Open Access Journal from MDPI, 14(8), 1161.

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Transcriptional Analysis in Diverse Tissues

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Total RNA was isolated from C2C12 cells, skeletal muscle, heart, adipose, kidney, and liver with RNA STAT-60 (Tel-Test Inc) as per manufacturer’s instructions. For tissues, RNA was isolated by disrupting tissue in RNA STAT-60 using 5 mm steel beads (Qiagen) and a TissueLyser II (Qiagen). RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR was performed using Power SYBR green (Applied Biosystems) and transcripts quantified on an ABI QuantiStudio 3 sequence detection system (Applied Biosystems). Data was normalized to 36B4 expression, unless otherwise noted, and results analyzed using the 2^−ΔΔCt method and reported as relative units to controls. Primer sequences are listed in Table S1.

, & Hecht M.H. (1994). De novo design of beta-sheet proteins.. Proceedings of the National Academy of Sciences of the United States of America, 91(19), 8729-8730.

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Quantification of Cytokines in Tissue Extracts

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Cytokines were quantified in tissue protein extracts and cell culture supernatants using the FlowCytomix kit (eBioscience, Vienna, Austria) according to the manufacturer's protocol. For quantification of cytokines in the ear tissue, ears were incubated in a protein extraction buffer containing 150 nM NaCl, 50 mM Tris and protease inhibitor cocktail (Roche Diagnostics, Rotkreuz, Switzerland). 5-mm steel beads (Qiagen, Hilden, Germany) were added to the samples, and the homogenization process was performed using a Qiagen tissue lyzer (4×1 min, 4°C, 30 Hz). Homogenized tissues were then centrifuged for 5 min at full speed and cytokines were quantified in the supernatant.

Aebischer D., Willrodt A.H, & Halin C. (2014). Oxazolone-Induced Contact Hypersensitivity Reduces Lymphatic Drainage but Enhances the Induction of Adaptive Immunity. PLoS ONE, 9(6), e99297.

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Gill Tissue RNA Extraction Protocol

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Gill samples stored on RNALater (approx. 20 mg tissue) were transferred to 0.5 ml of Qiazol lysis reagent (Qiagen, Germany) and homogenized in a TissueLyser II (Qiagen) using 5 mm steel beads (Qiagen). Chloroform (0.1 ml) was mixed into the homogenate followed by centrifugation. The top aquatic phase was collected, mixed with 70% ethanol (1:1), and transferred to an RNeasy filter tube for isolation of total RNA according to the RNeasy kit protocol (Qiagen). Total RNA was eluated in 50 μl RNase free water, RNA concentration and purity was measured in a NanoDrop 8000 spectrophotometer, and the sample was immediately stored at −80°C until microarray analyses.

Gjessing M.C., Krasnov A., Timmerhaus G., Brun S., Afanasyev S., Dale O.B, & Dahle M.K. (2020). The Atlantic Salmon Gill Transcriptome Response in a Natural Outbreak of Salmon Gill Pox Virus Infection Reveals New Biomarkers of Gill Pathology and Suppression of Mucosal Defense. Frontiers in Immunology, 11, 2154.

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mRNA Expression Profiling of Frozen Tumors

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For 80 patients, mRNA gene expression data were obtained through microarray analysis performed on a previous occasion (data not shown). Of these, 78 specimens were also available for IHC staining of SLC7A5. Briefly, freshly frozen primary tumor tissues (stored at −80°C) with confirmed tumor cell content were homogenized using a TissueLyser II (Qiagen AB) with 5 mm steel beads (Qiagen AB) for 2×2 min at 30 Hz. Lysing and RNA extraction was performed using the Allprep DNA/RNA/Protein mini kit (Qiagen AB). RNA concentrations were evaluated and purity determined according to the absorbance ratio at A260, A280 and A230 nm using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Values of A260/A280 between 1.8 and 2.1 and A260/A230>2.0 were considered as acceptable purity for microarray. RNA integrity number (RIN) values were determined using the Agilent RNA 6000 Nano Kit for the 2100 Bioanalyzer Instrument (Agilent Technologies, Inc.), with RIN>8 considered as an acceptable quality for subsequent microarray analysis. The RNA was stored at −80°C.

Törnroos R., Tina E, & Eremo A.G. (2021). SLC7A5 is linked to increased expression of genes related to proliferation and hypoxia in estrogen-receptor-positive breast cancer. Oncology Reports, 47(1), 17.

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RNA Isolation and cDNA Synthesis

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First, tissue samples were transferred from RNA later solution (Invitrogen, #AM7021) to RLT buffer (part of the RNEasy Mini Kit, Qiagen, Hilden, Germany, #74106) supplied with β‐mercaptoethanol and homogenized with 5‐mm steel beads (Qiagen; #69989) using TissueLyser II (#85300; Qiagen). Total RNA was isolated using RNEasy Mini Kit according to the manufacturer's instructions. The concentration and purity of isolated RNA were determined spectrophotometrically using a Nanodrop ND‐1000 spectrophotometer. The integrity of each RNA sample was analyzed using the Agilent RNA 6000 Nano kit run on an Agilent 2100 Bioanalyzer. Only samples without significant degradation were used for subsequent steps.
RNA was then reverse‐transcribed using M‐MLV (#28025013; Invitrogen) according to the manufacturer's instructions. Each 20 μL reaction contained up to 2 μg total RNA, 2.5 μm oligo(dT)₂₀ primers (#18418020; Invitrogen), 50 ng random hexamers (100 ng if more than 1 μg RNA was transcribed), 40 units of RNAseOUT, 200 units of M‐MLV reverse transcriptase, and other components as specified by the manufacturer.

Knedlík T., Vorlová B., Navrátil V., Tykvart J., Sedlák F., Vaculín Š., Franěk M., Šácha P, & Konvalinka J. (2017). Mouse glutamate carboxypeptidase II (GCPII) has a similar enzyme activity and inhibition profile but a different tissue distribution to human GCPII. FEBS Open Bio, 7(9), 1362-1378.

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Quantitative PCR Protocol for Gene Expression

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Slices were snap frozen in liquid nitrogen and stored at −80°C until RNA extraction. Total RNA was extracted with TRIzol Reagent (Thermofisher) according to the manufacturer’s manual. Samples were homogenized by crushing the tissue with 5 mm steel beads (Qiagen) with a Retsch MM 400 laboratory mill at 30 Hz for 2 min. Afterward, RNA was reverse transcribed into cDNA using the MLV Reverse Transcriptase (Promega). For qPCR, GoTaq qPCR Master Mix with BRYTE green (Promega) was used. qPCR was done using the Quantstudio3 real-time PCR system (Thermofisher). Gene-specific primers (Table 2) were produced by Integrated DNA Technologies (IDT Leuven). For analysis according to the Delta–Delta threshold (Ct) method, each Ct value was normalized against the respective reference genes (for mouse: the mean of Gapdh and Gtf2b; for human: the mean of GAPDH and YWHAZ). The best reference genes were selected out of six genes using GeNORM, performed in R environment. Each data point corresponds to one PCLS.

Dewyse L., De Smet V., Verhulst S., Eysackers N., Kunda R., Messaoudi N., Reynaert H, & van Grunsven L.A. (2022). Improved Precision-Cut Liver Slice Cultures for Testing Drug-Induced Liver Fibrosis. Frontiers in Medicine, 9, 862185.

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