After harvest, lung cells were lysed in TRIzol (Invitrogen). For lung tissues harvested from saline- or bleomycin-treated mice, samples were homogenized in TRIzol using the FastPrep-24 5G bead beating grinder and lysis system (MP Biomedicals). Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) and quantified using a nanodrop ND-100 spectrophotometer (Nanodrop Technologies). For each sample, an equal amount of RNA was converted to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems). qPCR was performed using Fast SYBR green master mix (Applied Biosystems) on a StepOne Real-Time PCR system (Applied Biosystems). Expression studies for human KLF4, ACTA2 (referred to in this manuscript as α-SMA), COL1α2, FOXM1, CCNB1 (referred to in this manuscript as CYCB1), APAF1, BIRC5, ECAD, MUC1, NCAD, VIM, and SNAIL1, as well as mouse Klf4, Acta2 (referred to in this manuscript as α-SMA), Col1α1, Ctgf, Tgf-β1, Mrc1, and Arg1, were performed using specific primers listed in Supplemental Table 1 . Relative quantification of gene expression was determined using the 2ΔCT method, and human GAPDH and mouse β-actin were used as reference genes for human and mouse samples, respectively. mRNA levels are presented as relative to the control value, set at 1.
Fastprep 24 5g bead beating grinder and lysis system
Manufactured by MP Biomedicals
Sourced in United States
The FastPrep-24 5G bead beating grinder and lysis system is a laboratory equipment designed for efficient sample homogenization and cell lysis. It utilizes high-speed agitation with ceramic or metal beads to disrupt a wide range of sample types, including tough plant and animal tissues, as well as microbial cells. The system is capable of processing multiple samples simultaneously, providing a standardized and reproducible method for sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 100 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by MP Biomedicals (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Turbo dnase, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Quantstudio 3, by Thermo Fisher Scientific (1 mentions)
Silica beads, by MP Biomedicals (1 mentions)
Iscript, by Bio-Rad (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Tripure rna isolation reagent, by Merck Group (1 mentions)
Sybr green kit, by Promega (1 mentions)
Cfx96 pcr machine, by Bio-Rad (1 mentions)
α sma, by Abcam (1 mentions)
Protease inhibitor, by Roche (1 mentions)
6 protocols using fastprep 24 5g bead beating grinder and lysis system
1
Quantitative Real-Time PCR Analysis
2
Isolating High-Quality RNA from Mouse Brain
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We obtained flash-frozen hippocampus, striatum, cerebellum, and cortex C57BL/6J mouse brain tissues from The Jackson Laboratory (JAX #000664, age = 20 weeks) from five male and five female mice. The samples arrived on dry ice, and we stored them at −70°C upon arrival. For each sample, we transferred ~30 mg of each brain region (or the entire brain region, in the case of hippocampus and striatum tissue) into an MP Biomedical Lysis D Matrix, 2ml tube (#6913500) containing 500μl of TRIzol reagent (Invitrogen #15596018) and lysed cells from each tissue on the FastPrep-24 5G bead beating grinder and lysis system (MP Biomedical #116005500). After lysis, we added 100μl of chloroform to the tube, centrifuged at 12,000×g for 15 minutes, and then transferred the clear top layer of the supernatant into a fresh tube. We next added an equivolume amount of isopropanol and centrifuged at 12,000×g for 10 minutes. We decanted the supernatant, washed the pellet twice with 75% ethanol, and resuspended the air-dried pellet in RNAse-free water. We incubated the final RNA product with TURBO DNase (Invitrogen #AM1907) for 30 minutes and assessed for RNA quality using a Qubit fluorometer and Agilent Fragment Analyzer. All RNA samples had an RNA quality number (RQN) score >7.
3
Quantitative PCR Analysis of Mouse Tissues
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Flash frozen perfused isolated mouse tissues (quadriceps, hippocampus) were placed in plastic tubes with silica beads (MP Biomedical, 116913100) and homogenized in the FastPrep-24 5G bead beating grinder and lysis system (MP Biomedical, 116005500) in TRI-zol (Invitrogen, 15596026). RNA was extracted via standard phenol-chloroform procedures followed by DNase digestion (ThermoFisher, AM2238). cDNA was generated using iScript (Bio-Rad Technologies, 1708891). Quantitative PCR was run using Sybrgreen (Life Technologies, 4309155) for chemical detection (Applied Biosystems; QuantStudio 3). Enrichment was determined based on double-delta CT value. Primers were ordered from IDT Predesigned qPCR Assays unless otherwise specified. Primers used are listed in the key resources table .
4
RNA Extraction and qPCR Analysis of Mouse sBAT
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RNA was isolated from frozen sBAT (approx. 5 mg) from mice of experiment 2 by lysing and homogenization using TriPure RNA Isolation Reagent (11667165001, Sigma Aldrich, Saint Louis, MO, USA) and a FastPrep-24™ 5G bead beating grinder and lysis system (4.0 m/s for 10 sec; MP Biomedicals™, Santa Ana, CA, USA). cDNA was synthesized from 1 μg RNA using M-MLV Reverse Transcriptase (M1705, Promega, Madison, WI, USA) and qPCR was conducted utilizing SYBR green kit (Promega, Madison, WI, USA) and a CFX96 PCR machine (Bio-Rad, Hercules, CA, USA), according to the manufacturers’ protocols. Gene expression was normalized to β-actin and expressed relative to the Young ZT0 group. Primer sequences are displayed in Supplementary Table 1 . Several mice were excluded due to technical errors.
5
Mouse and Human Lung Tissue Lysis
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50 mg of mouse lung tissue was weighed and put into a 2 mL reinforced tube with three stainless steel beads (MP Biomedicals, Irvine CA). 450 µL of lysis buffer containing 1 × protease inhibitor cocktail and 0.1% (v/v) of benzonase nuclease was added. Then, the tissue was homogenized in the MP Fastprep-24™ 5G bead beating grinder and lysis system (Irvine CA) at a 6 m/s speed for 40 s for 2 cycles. The resultant homogenate was chilled on ice for 5 min, and then centrifuged at 16,000 × g, 4 ℃ for 20 min. The supernatant was carefully transferred to a KingFisher 96 deep well plate. Human lung tissue sample was prepared as described above, except that the homogenization was performed at a speed of 6 m/s for 30 s for 4 cycles.
6
Protein Analysis of Cultured Cells and Tissues
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Cultured cells were directly lysed in RIPA buffer (Cell Signaling Technology) supplemented with protease inhibitors (Roche Diagnostics) and phosphatase inhibitor cocktail (EMD Biosciences). For lung tissues, samples were resuspended in RIPA buffer and homogenized using the FastPrep-24 5G bead beating grinder and lysis system (MP Biomedicals). Samples were then centrifuged at 12,000 rpm for 15 minutes to obtain clear lysates for subsequent protein studies. Sources of antibodies were as follows: KLF4 (catalog ab129473) and α-SMA (catalog ab7817), Abcam; COL1α2 (catalog PA5-50938l), Thermo Fisher Scientific; Fas (catalog 05-201) and FOXM1 (catalog ABE1000), MilliporeSigma; and CYCB1 (catalog 4135) and GAPDH-HRP conjugate (catalog 3683), Cell Signaling Technology. All antibodies were used on both human and mouse samples at a dilution of 1:1000 except GAPDH-HRP, which was used at a dilution of 1:3000. Protein quantification was performed by densitometric analysis of blots using ImageJ software (NIH). Protein levels are presented as relative to the control value, set at 1. Full, uncut gels for all the Western blots are shown in the supplemental material.
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