Zebrafish embryos were raised 30/well in a 6-well plate with 5 ml E3 medium, which was replaced once after hatching at 2 dpf. At 7 dpf, larvae were subjected to 10 min treatments of 250 mM NaCl (positive control), darkness, strobe light or ambient light (negative control). After treatments, each well was filled completely with ice-water, half of the volume in each well was removed (without sucking up fish) and the well again filled with ice-water. Larvae were transferred to pre-chilled 1.5 ml tube, 30 per tube, spun down for 5 sec and transferred back to ice. Water was removed down to 20 μl and fish were frozen using an ethanol/dry-ice bath. Samples were stored at −20 °C. For cortisol measurements, samples were thawed, combined with 150 μl H2O and homogenized for 20 sec with a pellet mixer (VWR International). 1 ml ethyl acetate (Sigma) was added per sample and tubes were vortexed at maximum speed for 30 sec. Solvent and aqueous phases were separated by centrifuging the tubes for 5 m at 3000 × g and 4 °C. Solvent (top) layer was transferred into a new 1.5 ml tube and evaporated at 25 °C for 18 h. Remaining cortisol was dissolved in 60 μl 0.2 % BSA in PBS and used directly in the Cortisol Saliva Luminescence Immunoassay Kit (IBL International) per the manufacturer’s instructions.
Pellet mixer
Manufactured by Avantor
Sourced in United States, Switzerland
The Pellet mixer is a laboratory equipment designed to effectively combine and homogenize various powdered or granular materials. It features a rotating drum or container that gently mixes the contents, ensuring uniform blending of the components.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Ethyl acetate, by Merck Group (2 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Centrifuge 5417r, by Eppendorf (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Anti phospho p65, by Cell Signaling Technology (1 mentions)
Anti mapk14, by Santa Cruz Biotechnology (1 mentions)
Anti myh11, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Agilent 4200 tapestation system, by Agilent Technologies (1 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Cfx manager 3, by Bio-Rad (1 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Icycler iq multicolor real time detection system, by Bio-Rad (1 mentions)
12 protocols using pellet mixer
1
Zebrafish Cortisol Stress Response
2
RNA Extraction from Tissue Samples
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RNA from 10–40 μg tissue was extracted using the TRIZOL reagent (Invitrogen, cat #15596026) as per manufacturer’s instructions. Muscle samples were snap frozen in liquid N2 and pulverized with a mortar. The pulverized tissue was resuspended in TRIZOL reagent and homogenized using a VWR Pellet Mixer (VWR, cat #431-0100) and disposable pestles (Argos Technologies Inc., cat #7339-901). Brain tissue was instead directly homogenized in TRIZOL reagent and processed as suggested by the manufacturer.
3
DNA Extraction and Transformant Identification
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DNA was extracted by adding 100 μL extraction buffer (100 mM Tris pH 8.0, 50 mM EDTA, 500 mM NaCl, 0.07% β-mercaptoethanol (v/v)) to the freshly harvested mycelium. The suspension was ground for 1 min using the VWR pellet mixer. 7 μL 20% (v/v) SDS and 26 μL 5 M KAc were added and the suspension was ground again for 1 min. The extraction samples were incubated for 10–60 minutes at 65°C followed by 10 minutes on ice. The samples were centrifuged for 10 minutes at 4°C at 16400 rpm using an Eppendorf Centrifuge 5417R, and the clear supernatant was transferred to a new tube. The centrifugation and transfer of supernatant was repeated. 128 μL ice-cold isopropanol and 12 μL 3 M NaAc were added to the samples, which were then incubated at −20°C for 10 minutes or longer. The samples were centrifuged for 5 minutes at 4°C at the maximal speed and the supernatant was discarded. The pellet was washed with 70% cold ethanol, air-dried and resuspended in MQ water to 100 μg mL-1.
To identify transformants that had integrated the mttA or mfsA gene, a PCR was carried out on the extracted DNA using primers specific for the mttA gene (Fw 5′CCC-GCA-AGT-ACA-GTA-AGA-ACG 3′ and Rv 5′CCT-GTA-CGG-AAC-CAG-ACT-CC 3′) and the mfsA gene (Fw 5′ TGA-TGG-GCT-CCT-TTA-ACT-GC 3′ and Rv 5′ GAT-AAG-ACC-GGC-GAT-AGT-GG 3′).
To identify transformants that had integrated the mttA or mfsA gene, a PCR was carried out on the extracted DNA using primers specific for the mttA gene (Fw 5′CCC-GCA-AGT-ACA-GTA-AGA-ACG 3′ and Rv 5′CCT-GTA-CGG-AAC-CAG-ACT-CC 3′) and the mfsA gene (Fw 5′ TGA-TGG-GCT-CCT-TTA-ACT-GC 3′ and Rv 5′ GAT-AAG-ACC-GGC-GAT-AGT-GG 3′).
4
Protein Extraction and Western Blot Analysis
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Freshly harvested tissues were snap frozen in liquid nitrogen, and then homogenized in liquid nitrogen with pellet mixer (VWR, 47747-370). Protein lysates were extracted from homogenized tissue or cultured SMCs by using an ice-cold cell lysis buffer (Cell Signaling, #9803) supplemented with a protease inhibitor cocktail (1%, Sigma, 539131). Protein concentration was measured with the BCA protein assay kit (Bio-Rad, 500). Equal amount of protein was resolved on SDS-PAGE gel for western blotting as described previously [9] (link). Antibodies used for western blot were as follows: anti-MAPK14 (1:1000, Santa Cruz, sc-535), anti-pMAPK14 (1:1000, Cell Signaling, #9211), anti-MYH11 (1:2000, Alfa Aesar, BT-562), anti-human CNN1 (1:1000, DAKO, M3556), anti-ACTA2 (1:1000, Sigma, A2547), anti-LMOD1 (1:2000, Proteintech, 15117-1-AP), anti-TAGLN (1:1000, Abcam, ab10135), anti-p65 (1:1000, Cell Signaling, #8242), anti-phospho-p65 (1:1000, Cell Signaling, #3033), and anti-TUBA (1:5000, Sigma, T5168).
5
RNA Extraction from Mouse Prefrontal Cortex
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Briefly, total RNA was extracted from the right PFC of the ketamine and saline-treated mice using the manufacturer’s protocol (RNeasy Mini Kit, Qiagen, Hilden, Germany). The tissue was ground and homogenized using a Pellet Mixer (VWR, Radnor, PA, USA). RNA concentration was assessed in the Nanodrop ND-2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and confirmed in the Agilent 4200 TapeStation System (Agilent, Santa Clara, CA, USA), which also revealed an RNA integrity number (RIN) above 8.0 for all samples.
6
Gene Expression Analysis in Mouse Tissues
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Tissue preparation and gene expression analysis was performed as done previously [33 (link)]. Wild-type male mice were either fasted overnight (12–15 h) with free access to water or fed ad libitum. Briefly, kidney, liver, and colon tissues were harvested from wild-type mice (28–30 weeks of age, N = 4–5) and snap frozen in liquid nitrogen until RNA extraction. Colon was collected as 5 cm of large intestine following the cecum. A VWR Pellet Mixer was used to homogenize each tissue on ice, and total RNA was isolated and purified using a RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Concentration, purity, and stability were confirmed via a NanoDrop 1000 and FlashGel™ System. First-strand cDNA synthesis was performed using SuperScriptTM III RT (Thermo Fisher Scientific) according to the manufacturer’s protocol, and concentration and purity was further confirmed using the NanoDrop 1000. For the quantitative real-time polymerase chain reaction (qRT-PCR) analysis, Taqman® gene expression assays were used for Slc16a5 (Thermo Fisher Scientific, Assay ID: Mm01252138) and the housekeeping gene Hprt (Assay ID: Mm03024075). Cycle threshold (Ct) were obtained from BioRad CFX Manager 3.0 software and imported into Excel for calculations.
7
RNA Quantification and Quality Assessment
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The concentration of RNA was measured with a Nanodrop 2000 UV-VIS spectrophotometer (Thermo Scientific, Wilmington, DE) and expressed as nanograms per microliter. The integrity of the RNA samples was assessed by sample preparation using Agilent RNA 6000 Nano Kits (Agilent Technologies, Santa Clara, CA) and algorithmic interpretation on the Agilent 2100 Bioanalyzer© (Agilent Technologies). RNA samples were stained with an intercalating dye, separated by microcapillary electrophoresis on gel-filled microfluidic chips, and detected by laser-induced fluorescence creating an electropherogram.32 (link) The Bioanalyzer compares ribosomal RNA fragments (18S and 28S) of the sample electropherograms to a software algorithm generating an RIN. The quality of ribosomal RNA, as reflected by an RIN value, is utilized as an alternate measurement of messenger RNA (mRNA) quality.32 (link) The pH was determined on a 10% (wt:vol) sample (200 mg) of prefrontal cortex homogenized with a VWR pellet mixer in 2 mL Milli-Q water.
8
Establishing Brain Tumor-Derived Cell Lines
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Intraoperative tumors were surgically resected during routine craniotomy and processed to establish BMCs as following: Tumor pieces were stored in physiological saline, 0.9% on ice until further processing. Following mincing using scalpels, the mechanically dissociated tissue was transferred to a 15 ml falcon tube containing trypsin/EDTA (0.05%) and incubated at 37 °C in a water bath for up to 20 minutes. In addition, the tissue was mechanically dissociated by usage of a Pellet Mixer (VWR International). The cell suspension was applied to a 70 µm cell strainer to remove undigested tissue fragments and cells in the flow-through were collected by centrifugation at 330 g for 5 min. Collected cells were resuspended in cell culture medium (DMEM, 4.5 g/L glucose, stabilized (GlutaMax) or conventional glutamine, pyruvate, 10% FBS, 1% P/S) and seeded on appropriate cell culture dishes. Cells were maintained for at least three days without medium change to achieve optimal recovering and attachment of tumor cells. BMCs were kept at low passages (2–20) and split according to their proliferative capacity (1:2–1:10) at a confluence of ~80%.
9
Zebrafish Cortisol Stress Response
10
Quantifying Influenza Virus in Mouse Lungs
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The right lung lobes of influenza-inoculated and comorbid mice were harvested 9–14 d after influenza inoculation (viral resolution phase). Lung lobes were finely sliced, and 30 mg of tissue per mouse was homogenized in 600 µl of RNeasy mini kit buffer RLT (QIAGEN) plus 100-fold–diluted β-mercaptoethanol using a pellet mixer (VWR). After centrifugation, the supernatant was taken and RNA was harvested with the RNeasy mini kit (QIAGEN). Then, RNA was reversed transcribed into cDNA using an iScript cDNA synthesis kit (Bio-Rad Laboratories). Influenza viral quantitation was achieved by quantitative PCR analysis. cDNA of influenza viral A matrix protein and β-actin (normalization control) was assayed with iQ SYBR green supermix (Bio-Rad Laboratories) with the indicated primers: influenza A matrix protein: forward, 5′-GACCAATCCTGTCACCTCTGAC-3′ and reverse, 5′-AGGGCATTTTGGACAAAGCGTCTA-3′; mouse β-actin: forward, 5′-TACAGCTTCACCACCACAGC-3′ and reverse, 5′-TCTCCAGGGAGGAAGAGGAT-3′. Samples were processed on an iCycler (Bio-Rad Laboratories), and cycle threshold (Ct) values were calculated with the iCycler iQ multicolor real-time detection system (version 3.1). Data were analyzed with the 2−ΔΔCt method, and values were normalized to β-actin transcription.
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