Omni bead ruptor 24 homogenizer, by Omni International - Product details

1

Quantitative Analysis of Mouse Brain Metabolites

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First, a homogenate of an intact mouse brain was prepared. The intact brain was thawed, the sample was placed in a homogenization tube, a 2-fold volume of water was added, homogenized using an Omni Bead Ruptor 24 homogenizer at a speed of 6.5 m/s for 45 s 2 times with an interval of 10 s.
For calibration and QC samples, 63 µL of intact tissue homogenate were taken into 1.5 mL test tubes, 7 µL of a 10-fold analyte standard solution in MeCN:H2O 1:1 was added, and mixed. The calibration range in brain homogenates was 1–1000 ng/mL.
A 2-fold volume of water was added to weighed samples of the brain of the mice and placed in test tubes for homogenization; homogenization was carried out using an Omni Bead Ruptor 24 homogenizer at a speed of 6.5 m/s 2 times for 45 s with an interval of 10 s.
The studied sample of the homogenate was obtained at a volume of 70 µL.
To all samples (calibration, QC and test), 280 μL of chilled MeCN with 50 ng/mL tolbutamide IS was added. Samples were vortexed for 10 s and kept at 4 °C for 15 min, then centrifuged for 10 min at 1500 g, and 150 μL of supernatants was obtained and transferred to a 96-well plate for HPLC-MS/MS analysis.

Zernov N., Veselovsky A.V., Poroikov V.V., Melentieva D., Bolshakova A, & Popugaeva E. (2022). New Positive TRPC6 Modulator Penetrates Blood–Brain Barrier, Eliminates Synaptic Deficiency and Restores Memory Deficit in 5xFAD Mice. International Journal of Molecular Sciences, 23(21), 13552.

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2

Quantifying Paclitaxel Levels in Tissue

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Blood samples (0.3 mL) and tumor tissue samples were collected at 0.2, 0.5, 1, 2, 4, 8, 12, and 24 h after PTX treatment. Tissues were homogenized by Omni Bead Ruptor 24 Homogenizer (Kennesaw, GA, USA). To quantify the PTX concentration in samples, solid phase extraction was applied before the measurement. PTX concentration was detected by liquid chromatography/mass spectrometry (LC/MS).

Chen S., Bian H, & Duan J. (2022). High-Intensity Focused Ultrasound Enhanced Anti-Tumor Activities of Paclitaxel in Breast Cancer in vitro and in vivo. Cancer Management and Research, 14, 1303-1312.

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3

Free Glycan Purification and Labeling

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Tissues were homogenized, in water, with 1.4 mm ceramic beads using an Omni Bead Ruptor 24 Homogenizer (Omni International, Kennesaw, GA, USA) and debris was removed by centrifugation at 14,000 rpm and 8 °C for 20 min. The protein concentration of clarified extracts was determined by BCA assay. An amount of sample equivalent to 240 μg of protein, for tissues, or 100 μL, for urine, were diluted to 1 mL with water and subjected to SPE purification using a Hypersep Hypercarb PGE SPE cartridge following the manufacturer's instructions and dried by centrifugal evaporation. The purified free glycans were labeled with aniline by reductive amination as previously described [23 (link),24 (link)] (15 μL aniline, 15 μL 1 M NaCNBH₃ in 70:30 DMSO:HOAc) at 37 °C for 18–24 h. After labeling the remaining aniline was removed by centrifugal evaporation at 37 °C. Samples were dissolved in water prior to analysis.

Lawrence R., Van Vleet J.L., Mangini L., Harris A., Martin N., Clark W., Chandriani S., LeBowitz J.H., Giugliani R., d'Azzo A., Yogalingam G, & Crawford B.E. (2019). Characterization of glycan substrates accumulating in GM1 Gangliosidosis. Molecular Genetics and Metabolism Reports, 21, 100524.

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4

Brain Metabolite Extraction Protocol

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Brain samples were homogenized in water with 1.4 mm ceramic beads using an Omni Bead Ruptor 24 Homogenizer (Omni International, Kennesaw, GA, USA). Protein concentration of the homogenate was determined using BCA assay. All homogenates were diluted with water to 4 μg protein/μL homogenate. An amount of sample equal to 200 μg of protein was extracted with 95/5 methanol/glacial acetic acid (v/v). Samples were filtered through 10 kDa centrifugal filter tubes and stored at −20 °C until use.

Lawrence R., Van Vleet J.L., Mangini L., Harris A., Martin N., Clark W., Chandriani S., LeBowitz J.H., Giugliani R., d'Azzo A., Yogalingam G, & Crawford B.E. (2019). Characterization of glycan substrates accumulating in GM1 Gangliosidosis. Molecular Genetics and Metabolism Reports, 21, 100524.

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5

Traumatic Brain Injury Induction in Drosophila

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The TBI methods used for Drosophila have been previously detailed [10 (link)]. Briefly, flies from different genders, ages, or treatment regimens were anesthetized and placed in 2 mL screw cap tubes (10 flies/tube) [9 (link)]. Control and traumatized flies were allowed to recover from anesthesia before being placed in the Omni Bead Ruptor-24 homogenizer (Omni International, Kennesaw, GA, USA) and subjected to specific pre-programmed shaking/trauma conditions involving 5 s trauma bouts [9 (link)]. Following the completion of the injury, all fly cohorts are returned to vials containing control or treated media, allowed to recover, and maintained using standard care and culturing conditions [9 (link)]. For this study, fly cohorts were exposed to 10 mild bouts of trauma (mTBI; 10×, 2.1 m/s) or one severe trauma bout (mTBI; 1×, 4.3 m/s) [9 (link)].

Molina B., Mastroianni J., Suarez E., Soni B., Forsberg E, & Finley K. (2021). Treatment with Bacterial Biologics Promotes Healthy Aging and Traumatic Brain Injury Responses in Adult Drosophila, Modeling the Gut–Brain Axis and Inflammation Responses. Cells, 10(4), 900.

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6

Gene Expression Analysis in Rgs2/5 dbKO Mice

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Total RNA was extracted from heart apexes of male and female WT and Rgs2/5 dbKO mice via a Trizol extraction method (ThermoFisher Scientific) and utilizing an Omni Bead Ruptor 24 homogenizer (Omni International) with tissue homogenizer tubes. RNA was purified using the Purelink RNA Mini Kit (ThermoFisher) and then reverse-transcribed using a Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific), all according to the manufacturer’s instructions. The primer probes listed in Table S4 were used in the real-time PCR assays with TaqMan gene expression master mix (ThermoFisher Scientific): The ΔC_t method (where C_t is threshold cycle) was used to calculate mRNA expression after normalization to Gapdh expression.

Dahlen S.A., Bernadyn T.F., Dixon A.J., Sun B., Xia J., Owens E.A, & Osei-Owusu P. (2022). Dual loss of regulator of G protein signaling 2 and 5 exacerbates ventricular myocyte arrhythmias and disrupts the fine-tuning of Gi/o signaling. Journal of molecular and cellular cardiology, 170, 34-46.

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7

Colon Epithelium Metabolomic Analysis

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Metabolomic analysis was performed using gas chromatography-mass spectrometry (GC-MS) as previously described with slight modifications [20 (link)]. Namely, ~ 5–10 mg of distal colon epithelium or tumors were washed twice in ice-cold PBS; then, metabolites were extracted with 800 μL of 90% cold methanol containing 2 μg/mL of succinic acid-2,2,3,3-d4 as an internal standard using the Omni Bead Ruptor 24 homogenizer (Omni International), and dried with a Speed-Vac. Dried extracts were derivatized with O-methoxylamine hydrochloride and N-methyl-N-trimethylsilyltrifluoracetamide containing 1% trimethylchlorosilane, respectively. Derivatized samples were analyzed in an Agilent 6890 GC-5973iMS equipment with a 30-m Phenomex ZB5-5 MSi column with a 5-m long guard column. Raw data were normalized with the internal standard and tissue mass.

Maiuri A.R., Li H., Stein B.D., Tennessen J.M, & O’Hagan H.M. (2018). Inflammation-induced DNA methylation of DNA polymerase gamma alters the metabolic profile of colon tumors. Cancer & Metabolism, 6, 9.

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8

Gene Expression Analysis in Rgs2/5 dbKO Mice

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Total RNA was extracted from heart apexes of male and female WT and Rgs2/5 dbKO mice via a Trizol extraction method (ThermoFisher Scientific) and utilizing an Omni Bead Ruptor 24 homogenizer (Omni International) with tissue homogenizer tubes. RNA was purified using the Purelink RNA Mini Kit (ThermoFisher) and then reverse-transcribed using a Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific), all according to the manufacturer’s instructions. The primer probes listed in Table S4 were used in the real-time PCR assays with TaqMan gene expression master mix (ThermoFisher Scientific): The ΔC_t method (where C_t is threshold cycle) was used to calculate mRNA expression after normalization to Gapdh expression.

Dahlen S.A., Bernadyn T.F., Dixon A.J., Sun B., Xia J., Owens E.A, & Osei-Owusu P. (2022). Dual loss of regulator of G protein signaling 2 and 5 exacerbates ventricular myocyte arrhythmias and disrupts the fine-tuning of Gi/o signaling. Journal of molecular and cellular cardiology, 170, 34-46.

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9

Traumatic Brain Injury in Drosophila

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Flies were incapacitated using CO₂ and placed in a clean 2-ml screw cap tube (10 flies/tube). To eliminate any confounding effects of CO₂ exposure and the stress of being placed in a 2-ml tube, both control (non-injured) and TBI treated fly cohorts were subjected to the above conditions. For injury, tubes containing flies were placed into the Omni Bead Ruptor-24 homogenizer (Omni International, Kennesaw, GA, USA) and subjected to specific pre-programmed shaking conditions. This instrument permits highly controlled shaking conditions that include a broad range of intensities (0.8–8.0 m/s range). Flies were subjected to a particular intensity (m/s) injury for 5 seconds. For multi-bout conditions, fly cohorts were injured (5 seconds) and allowed to recover for 30 seconds before the start of subsequent injury bouts. Following the completion of the injury, all fly cohorts were put into vials, which were placed on their sides to allow for a full recovery.

Barekat A., Gonzalez A., Mauntz R.E., Kotzebue R.W., Molina B., El-Mecharrafie N., Conner C.J., Garza S., Melkani G.C., Joiner W.J., Lipinski M.M., Finley K.D, & Ratliff E.P. (2016). Using Drosophila as an integrated model to study mild repetitive traumatic brain injury. Scientific Reports, 6, 25252.

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10

Quantitative Analysis of RGS Genes

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Expression levels of RGS2, RGS4, and RGS5 were determined by real‐time PCR using RNA extracted from uterine arteries of NP, D10, D15, D18, and PPD3 WT, Rgs2^−/−, and Rgs5^−/− mice and from mesenteric arteries and aorta of NP, D15, and PPD3 WT, Rgs2^−/−, and Rgs5^−/− mice. Total RNA was isolated using the TRIzol extraction method (Thermo Fisher Scientific, Waltham, MA), with tissue homogenizer tubes and an Omni Bead Ruptor 24 homogenizer (Omni International, Kennesaw, GA). RNA was purified using the Purelink RNA Mini Kit (Thermo Fisher Scientific). RNA was then reverse‐transcribed into cDNA using a Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. The following primer probes were used in the real‐time PCR assays with TaqMan gene expression master mix (Thermo Fisher Scientific), as directed by the manufacturer: Rgs2, Mm01292909_g1; Rgs5, Mm00654112_m1; Rgs4, Mm00501389_m1; and Gapdh, Mm99999915_g1. The ∆C_t method (where C_t is threshold cycle) was used to calculate RGS2, RGS4, and RGS5 mRNA expression after normalization to Gapdh expression.

Koch J.N., Dahlen S.A., Owens E.A, & Osei‐Owusu P. (2019). Regulator of G Protein Signaling 2 Facilitates Uterine Artery Adaptation During Pregnancy in Mice. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(9), e010917.

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Omni bead ruptor 24 homogenizer

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10 protocols using omni bead ruptor 24 homogenizer

Quantitative Analysis of Mouse Brain Metabolites

Quantifying Paclitaxel Levels in Tissue

Free Glycan Purification and Labeling

Brain Metabolite Extraction Protocol

Traumatic Brain Injury Induction in Drosophila

Gene Expression Analysis in Rgs2/5 dbKO Mice

Colon Epithelium Metabolomic Analysis

Gene Expression Analysis in Rgs2/5 dbKO Mice

Traumatic Brain Injury in Drosophila

Quantitative Analysis of RGS Genes

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