Fastprep 24 instrument

Manufactured by MP Biomedicals

Sourced in United States, France, Germany, United Kingdom

The FastPrep-24 Instrument is a high-speed benchtop sample homogenizer designed for efficient lysis and disruption of a variety of sample types, including tissue, cells, and microorganisms. The instrument utilizes rapid, high-speed agitation to effectively break down samples, preparing them for subsequent analysis or processing.

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Lab products found in correlation

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FastPrep-24 (832 citations) FastPrep (251 citations) FastPrep Instrument (116 citations) MP FastPrep-24 Instrument (10 citations) Fastprep-24 bead-beating instrument (4 citations) FastPrep-24TM instrument (3 citations) FastPrep-24TM 5G Instrument (3 citations) FastPrep Instrument FP-24 (2 citations) FastPrep-24 Bead-Beater Instrument (2 citations) Fastprep-24 Sample Preparation Instrument (2 citations)

239 protocols using fastprep 24 instrument

Saliva and Stool DNA Extraction

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All subjects underwent stool and saliva collection on the same day. Unstimulated saliva samples collected from subjects were immediately stored at -20°C until use. The saliva samples were homogenized with zirconia beads in a 2.0-mL screw cap tube by FastPrep 24 Instrument (MP Biomedicals, Santa Ana, CA) at 5 m/s for 90 sec. DNAs were extracted from 100 μL of the saliva and purified with the MORA-EXTRACT DNA extraction kit (Kyokuto Pharmaceuticals, Tokyo, Japan) in accordance with the manufacturer's instructions. The DNAs were eluted with 100 μL of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
Fecal samples were immediately suspended in a solution containing 100 mM Tris-HCl (pH 9.0), 40 mM Tris-EDTA (pH 8.0), 4 M guanidine thiocyanate, and 0.001% bromothymol blue. An aliquot of 1.2 mL of the suspension was homogenized with zirconia beads in a 2.0-ml screw cap tube by FastPrep 24 Instrument (MP Biomedicals) at 5 m/s for 2 min and placed on ice for 1 min. After centrifugation at 5000 × g for 1 min, DNA was extracted from 200 μL of the suspension using an automatic nucleic acid extractor (Precision System Science, Chiba, Japan). MagDEA DNA 200 (GC) (Precision System Science) was used as the reagent for automatic nucleic acid extraction [27 (link), 28 (link)].

Abe K., Takahashi A., Fujita M., Imaizumi H., Hayashi M., Okai K, & Ohira H. (2018). Dysbiosis of oral microbiota and its association with salivary immunological biomarkers in autoimmune liver disease. PLoS ONE, 13(7), e0198757.

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Isolation and Purification of Intestinal Epithelial and Lamina Propria Leukocytes

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Isolation of lamina propria leukocytes was performed as described previously⁶¹. In brief, intestines were isolated, cut open longitudinally and washed briefly in PBS. Dissociation of epithelial cells was performed by shaking at 100 rpm/min at 37°C in HBSS containing 5 mM EDTA and 10 mM Hepes for 20 min. Epithelial cells were collected and spun down for 5 min at 2000 rpm/min at 4°C and cell pellets were resuspended in Trizol using a FastPrep^®-24 Instrument (MP Biomedicals). To obtain highly purified epithelial cells, the cell pellets were digested for 20 min using Dispase (5 U/ml; BD), Collagenase D (0.5 mg/ml; Roche) and DNaseA (0.5 mg/ml; Sigma-Aldrich). Cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI), CD45 and EpCAM antibodies and sorted using a BD FACSAria III cell sorter (BD Biosciences). The remaining lamina propria fraction was cut into pieces of 1 mm² before performing enzymatic digestion using the same enzyme mix as for the digestion of epithelial cells. Lymphocyte enrichment was performed by Percoll gradient centrifugation (Sigma-Aldrich).

Hernández P.P., Mahlakoiv T., Yang I., Schwierzeck V., Nguyen N., Guendel F., Gronke K., Ryffel B., Hoelscher C., Dumoutier L., Renauld J.C., Suerbaum S., Staeheli P, & Diefenbach A. (2015). Interferon-λ and interleukin-22 cooperate for the induction of interferon-stimulated genes and control of rotavirus infection. Nature immunology, 16(7), 698-707.

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Lipid Extraction from Oil Bodies

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The lipids from the oil bodies were extracted by adding 500 µL of isooctane into 0.2 g of oil bodies that were previously dried in a vacuum oven at 40 °C for 48 h. The tubes were homo-genised into a FastPrep®-24 Instrument (MP Biomedicals, Cambridge, UK) for 30 s and microcentrifuged (MSE Micro Centaur, Sanyo, London, UK) at 13 000g for 5 min. A known volume of the supernatant was pipetted into labelled Eppendorf tubes. This extraction step was repeated 3 times. One mL of the pooled supernatant was poured into a 10 mL glass bijou bottle and dried in a vacuum oven for a few hours; this fraction contained the lipids. The isooctane remaining in the tubes was evaporated in vacuo and the pellet stored in a freezer at -20 °C for protein analysis. The lipid content was determined gravimetrically by calculating the difference in the sample weight measured before and after the extraction process.

Grundy M.M., Carrière F., Mackie A.R., Gray D.A., Butterworth P.J, & Ellis P.R. (2016). The role of plant cell wall encapsulation and porosity in regulating lipolysis during the digestion of almond seeds. Food & function, 7(1).

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Transcriptome Analysis of Mn-Treated Samples

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Total RNA of samples Mn-C, Mn-CC, Mn-9OHAD, Mn-ADD, and Mn-BNA were isolated using the FastRNA™ Pro Blue Kit and the Fast Prep-24™ Instrument (MP Biomedicals, SantaAna, CA). NanoDrop (NanoDrop Technologies,Wilmington, DE) was used to verify the concentration and quality of each RNA sample. Total RNA was incubated with Rnasefree DNase Set (Qiagen) to remove DNA contamination according to the manufacturer’s instructions. Messenger RNA (mRNA) was further purified by depleting ribosomal RNA (rRNA) and tRNA with Terminator™ 5′-phosphate dependent exonuclease (Epicenter, Madison, WI) digestion. Double-stranded cDNA was synthesized using a kit obtained from Beijing Dingguo Chang sheng Biotechnology Co., Ltd., (Beijing, China). RNA sequencing was conducted with an Illumina Genome Analyzer IIx according to the manufacturers’ protocols.

Liu M., Zhu Z.T., Tao X.Y., Wang F.Q, & Wei D.Z. (2016). RNA-Seq analysis uncovers non-coding small RNA system of Mycobacterium neoaurum in the metabolism of sterols to accumulate steroid intermediates. Microbial Cell Factories, 15, 64.

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Bacterial Proteome Extraction and Quantification

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Preinocula were grown in 5 ml TSBg at 37°C and 200 rpm overnight (ON). Bacterial concentrations were estimated by measuring their optical density (OD₆₀₀). Normalized bacterial aliquots were diluted 1:50 in 250 ml Erlenmeyer flasks containing 50 ml of TSBg. Cultures were incubated at 37°C and 200 rpm and samples extracted at the experiment given time points. Samples were centrifuged for 3 min at 4,400 g and 4°C, pellets frozen in liquid nitrogen and stored at –80°C until required. Pellets were thawed, washed once with phosphate buffered saline (PBS) and resuspended in 1 ml of buffer containing 7 M urea, 2 M thiourea, 2% CHAPS and 50 mM dithiothreitol (DTT) or 1 ml of PBS for LC–MS-based proteomics or Western blot experiments, respectively. Resuspended bacteria were transferred to Fast Prep tubes containing 100 μm acid-washed glass beads (Sigma) and mechanically lysed in a FastPrep-24 instrument (MP Biomedicals) for 45 s and speed 6, twice. Tubes were centrifuged for 10 min at 21,000 g and 4°C. Supernatants, containing total protein extracts, were quantified using Bradford protein assay kit (Bio-Rad) and samples prepared at the desired concentration in Laemmli buffer.

Caballero C.J., Menendez-Gil P., Catalan-Moreno A., Vergara-Irigaray M., García B., Segura V., Irurzun N., Villanueva M., Ruiz de los Mozos I., Solano C., Lasa I, & Toledo-Arana A. (2018). The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus. Nucleic Acids Research, 46(3), 1345-1361.

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Rhizosphere RNA Extraction Protocol

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RNA was extracted using a protocol adapted from two previously described protocols (Griffiths et al., 2000; Töwe et al., 2011). Briefly, 0.4 g rhizosphere sample were mixed in a bead tube (PowerBead Pro Tubes QIAGEN®) with equal amounts of Hexadecyltrimethylammonium bromide (CTAB; 10% (wt/vol)) extraction buffer (240 mM potassium phosphate, 0.7 M NaCl, 10 μl ml⁻¹ ß-mercaptoethanol, pH 8) and phenol-isoamyl-chlorophorm with volumetric ratio of 25:24:1). Homogenization was performed twice for 40 s at a speed of 5.5 m s⁻¹ in a bead mill (MP Biomedicals™ FastPrep-24 Instrument) interrupted by inverting the tubes for two minutes to avoid heat degradation. Followed by centrifugation for (20 min, 4 °C 16,100×g). Resulting aqueous raw extract was recovered and purified twice with chloroform:isoamylalcohol (with volumetric ratio of 24:1). Subsequently, RNA was precipitated with 10% polyethyleneglycol 6000 while being incubated for 90 min on ice. The precipitate was recovered by centrifugation (30 min, 4 °C, 16,100×g) and purified by washing with ice cold 70% ethanol. Subsequent to centrifugation (5 min, 4 °C, 16,100×g) the RNA pellet was suspended in 80 µl RNAse-free water.

Lewin S., Francioli D., Ulrich A, & Kolb S. (2021). Crop host signatures reflected by co-association patterns of keystone Bacteria in the rhizosphere microbiota. Environmental Microbiome, 16, 18.

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Protein Extraction and Quantification Protocol

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Cell pellets were harvested from 50 mL of each culture and resuspended in 200 µL of protein extraction buffer (1X AEBSF, Roche and 1X PSB, Agrisera). Samples were then homogenized using the CY:24×2 rotor of the FastPrep-24 Instrument (MPBIO) for two 1 min periods with 1 min of cooling on ice after each treatment. The protein extracts were then centrifuged at 10,000×g for 3 min to remove insoluble material and unbroken cells. Protein concentrations of extracts were quantified using the microplate DC protein assay (Bio-Rad) with bovine gamma globulin (BGG) as a comparative protein standard.
The sample preparation, SDS-PAGE, immunoblotting and quantitation protocols were followed according to those described previously [26] (link). Immunoblotting of PSHCP required a shorter transfer of 45 min. onto nitrocellulose membranes. Primary antibodies (Agrisera) were diluted in 2% ECL Advance blocking agent (GE Healthcare) in TBS-T, RbcL 1∶10,000 and PSHCP 1∶2,000. HRP-conjugated goat anti-rabbit (Agrisera) was employed as the secondary antibody at a dilution of 1∶40,000. Immunoblots were developed with ECL Advance (GE Healthcare) and images were captured on a VersaDoc Imager (BioRad) and analyzed using Quantity One and Image Lab 3.0 software (BioRad).

Whidden C.E., DeZeeuw K.G., Zorz J.K., Joy A.P., Barnett D.A., Johnson M.S., Zhaxybayeva O, & Cockshutt A.M. (2014). Quantitative and Functional Characterization of the Hyper-Conserved Protein of Prochlorococcus and Marine Synechococcus. PLoS ONE, 9(10), e109327.

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Lipidomic analysis of ZIKV-infected tissues

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Five-day mock- or ZIKV-infected tissues were snap-frozen in liquid nitrogen and stored until extraction. Frozen tissue samples were crushed with a FastPrep^®−24 Instrument (MP Biomedical, Santa Ana, CA) in 1 mL HBSS (Invitrogen, 14170112). After 2 crush cycles (6.5 m/s, 30 s), 50 µL were withdrawn for protein quantification. Different classes of lipids were extracted according to the manufacturer’s instructions. Signal intensity for specific lipid species was extracted according to the retention time. Samples with spiked internal standards were analyzed by either gas/liquid chromatography followed by mass spectrometry as described in Supplementary methods. Representative chromatogram of different lipid species have been provided in Supplementary Figs. 8–10. The Mass Spectrometry conditions of different lipid species have been indicated in Supplementary Tables 2–5.

Chen Q., Gouilly J., Ferrat Y.J., Espino A., Glaziou Q., Cartron G., El Costa H., Al-Daccak R, & Jabrane-Ferrat N. (2020). Metabolic reprogramming by Zika virus provokes inflammation in human placenta. Nature Communications, 11, 2967.

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RNA Extraction from Lip Tissues

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Total RNA was extracted from 15 lip tissue samples (five replicates per lip region) from G. permaxillaris and ten lip tissue samples from G. bellcrossi using the ReliaPrep™ RNA Tissue Miniprep System Kit (Promega). Each sample consisted of epidermis, dermis and the underlying soft connective tissues in the specified lip regions (Fig. 1). Tissue samples were placed into tubes containing 250 µl of lysis buffer mixed with 1-Thioglycerol and 1.4 mm ceramic beads. The tissues were homogenized thoroughly by FastPrep-24 Instrument (MP Biomedicals, CA, USA) and RNA extraction was performed based on manufacturer's ReliaPrep™ protocol for fibrous tissues. The extraction protocol followed included a column-based genomic DNA removal step and several purification steps. The total RNAs were diluted in 50 µl nuclease-free water and were quantified by a Nanophotometer (IMPLEN GmbH, Munich, Germany). The qualities of total RNAs were measured by R6K ScreenTape System using an Agilent 2200 TapeStation (Agilent Technologies), and all samples had a RNA integrity number (RIN) minimum of eight. Around 200 ng of the extracted total RNA from each sample was used for cDNA synthesis based on the manufacturer's protocol of High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and 1:4 times cDNA dilution was used as input to perform qPCR.

Lecaudey L.A., Singh R., Sturmbauer C., Duenser A., Gessl W., & Ahi E.P. (2021). Transcriptomics Unravels Molecular Players Shaping Dorsal Lip Hypertrophy in the Vacuum Cleaner Cichlid, Gnathochromis Permaxillaris.

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Quantifying Gene Expression with qPCR

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The whole seedlings from three independent wells were immediately frozen in liquid nitrogen. The tissue was homogenized in tubes with 1 g of 1.3 mm silica beads using a FastPrep-24 instrument (MP Biomedicals, USA). Total RNA was isolated using a Spectrum Plant Total RNA kit (Sigma-Aldrich, USA) and treated with a DNA-free kit (Ambion, USA). Subsequently, 1 μg of RNA was converted into cDNA with M-MLV RNase H⁻ Point Mutant reverse transcriptase (Promega Corp., USA) and an anchored oligo dT21 primer (Metabion, Germany). Gene expression was quantified by q-PCR using a LightCycler 480 SYBR Green I Master kit and LightCycler 480 (Roche, Switzerland). The PCR conditions were 95 °C for 10 min followed by 45 cycles of 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 20 s. Melting curve analysis was then conducted. Relative expression was normalized to the housekeeping genes AtSAND and BnTIP41. Primers were designed using PerlPrimer v1.1.21^{39 (link)}. A list of the analysed genes and primers is available in Table S2.

Leontovyčová H., Kalachova T., Trdá L., Pospíchalová R., Lamparová L., Dobrev P.I., Malínská K., Burketová L., Valentová O, & Janda M. (2019). Actin depolymerization is able to increase plant resistance against pathogens via activation of salicylic acid signalling pathway. Scientific Reports, 9, 10397.

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