Primary hippocampal and cortical neurons were prepared from day 18 embryos of Sprague-Dawley rats as described previously77 (link). In brief, hippocampal tissues were separately dissociated by gentle trituration in a calcium-free Hank’s balanced salt solution and centrifuged at 1000×g. Cells were resuspended in Neurobasal media (Gibco) containing B27 supplement (Thermo Fisher Scientific), 1% antibiotic/antimitotic solution (104 Unit of penicillin G/ml, 10 mg streptomycin/ml and 25 μg amphotericin B/ml) (Sigma), and plated at a density of 40,000 cells/ml in 96-well plates (Corning) coated with polyethyleneimine (Sigma) or 160,000 cells/ml in multichannel electrode array chambers (Harvard Apparatus) coated with polyethyleneimine (Sigma) and laminin (Sigma). Three hours after plating the media was replaced with serum-free Neurobasal medium containing 1% B-27 supplement (Gibco). Immunofluorescent staining showed that cultures were >98% MAP-2+ neurons. Hippocampal cultures were used after 3 days of culture in vitro (DIV) as developing neurons, and 14 DIV as mature neurons. All animal procedures were approved by the Johns Hopkins University Animal Care and Use Committee.
Polyethyleneimine
Manufactured by Merck Group
Sourced in United States, Germany, Poland, India, France, China, Sao Tome and Principe, United Kingdom
Polyethyleneimine is a water-soluble, cationic polymer that is commonly used as a transfection agent in molecular biology and cell culture applications. It functions as a non-viral vector for the delivery of nucleic acids into eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal medium, by Thermo Fisher Scientific (9 mentions)
Glutaraldehyde, by Merck Group (8 mentions)
Sodium hydroxide (naoh), by Merck Group (8 mentions)
Sodium chloride (nacl), by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
B27 supplement, by Thermo Fisher Scientific (8 mentions)
Hydrochloric acid (hcl), by Merck Group (7 mentions)
Polybrene, by Merck Group (6 mentions)
Trypsin, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Merck Group (6 mentions)
Polyvinylpyrrolidone, by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (5 mentions)
Acetone, by Merck Group (4 mentions)
Phosphate buffered saline (pbs), by Merck Group (4 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions)
Laminin, by Merck Group (4 mentions)
Laminin, by Thermo Fisher Scientific (4 mentions)
Paraformaldehyde (pfa), by Merck Group (3 mentions)
229 protocols using polyethyleneimine
1
Isolation and Culture of Rat Hippocampal and Cortical Neurons
2
Lentivirus and Retrovirus Production
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The lentivirus plasmids and another two packaging plasmid (psPAX2 and pMD2.G) were transduced to HEK-293T cells by using polyethyleneimine (Sigma-Aldrich, St. Louis, MO, USA). After 48 and 72 h, the supernatant containing lentivirus was harvested and filtered by using 0.45-μm syringe filter. The retrovirus plasmid was transduced to Platinum-E cells by using polyethyleneimine (Sigma-Aldrich, St. Louis, MO, USA). After 48, the supernatant containing retrovirus was harvested and filtered by using 0.45-μm syringe filter.
3
Lentiviral Transduction of EL4 Cells
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293 FT cells were seeded in a 15-cm dish in 20 mL of complete DMEM medium. Cells were incubated for 24 hours at 37°C. Full culture medium was exchanged with 8.1 mL Opti-MEM per dish. 293 FT cells were transfected with the application of polyethyleneimine (764582, Merck Life Science, Shanghai, China). The dish was mixed by rocking gently to spread the DNA. Three days after transfection, the medium containing cell debris was centrifuged and concentrate virus were collected. EL4 cells were seeded and put in 200 uL lentivirus stock. The plate was swirled to mix and incubated overnight. Three days later, GFP-positive cells were sorted and incubated at 37°C in a CO2 incubator.
4
Lentiviral Knockdown and Overexpression of Dicer
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The pLKO.1-puro-based lentiviral vectors TRCN0000290426 (shDicer#1), and TRCN0000290489 (shDicer#2), and the control plasmids TRC025.shLKO (shCtrl#1), and TRC2. Scramble (shCtrl#2), were purchased from the National RNAi Core Facility at Academia Sinica, Taipei, Taiwan. HEK293T cells were cotransfected with the lentivirus expression plasmid, packaging plasmid (pCMV-dR8.91), and envelope plasmid (VSV-G expressing plasmid, pMD2.G) with polyethyleneimine (Merck) for 48 h. We generated a recombinant lentivirus from the culture medium. The cells were infected with lentiviruses combined with 8 μg/mL polybrene, and stable cells were selected using 0.5–1 μg/mL puromycin.
To overexpress Dicer and phosphomimetic Dicer, the Dicer WT plasmid (pCAGGS-Flag-hsDicer plasmid #41584) was purchased from Addgen, and then Dicer was subcloned into the pcDNA6/myc-His vector. The pcDNA6-Dicer S1016A and S1016E mutations were achieved using the Q5 site-directed mutagenesis kit (NEB E0554S) and specific primers (Supplementary Table 1 ). Cells were transfected with a Dicer WT, S1016A, or S1016E plasmid or a control vector (pcDNA6) for 48 h by using the DreamFect Gold transfection reagent (DG80500, OZ Biosciences, France) in accordance with the manufacturer's instructions, and stable cell lines were selected using 5 μg/mL blasticidin (ant-bl-05; InvivoGen, USA).
To overexpress Dicer and phosphomimetic Dicer, the Dicer WT plasmid (pCAGGS-Flag-hsDicer plasmid #41584) was purchased from Addgen, and then Dicer was subcloned into the pcDNA6/myc-His vector. The pcDNA6-Dicer S1016A and S1016E mutations were achieved using the Q5 site-directed mutagenesis kit (NEB E0554S) and specific primers (
5
Fluorescence Imaging of Photosensitizer Uptake
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For fluorescence microscopy, U251 cells were cultured on 8-chamber glass-bottomed dishes coated with 0.04% Polyethyleneimine (Merck, Darmstadt, Germany) at 3 × 104 cells/well. Cells were cultured for 24 h before the transport assay. Precooled (4 °C) or prewarmed (37 °C) fresh HEPES containing DMEM without phenol red (FUJIFILM Wako Pure Chemical, Osaka, Japan) with 2 µM photosensitizer was applied to the culture for 30 min at 4 or 37 °C. Cells were washed twice with PBS and fixed with 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer for 30 min at room temperature. Fluorescence of the photosensitizer was observed under an Eclipse Ti-U inverted microscope (Nikon, Tokyo, Japan) equipped with a filter cube (Ex: 340–380 nm; DM: 400 nm; Em: 672–716 nm) and CMOS Zyla5.5 camera (Andor technology, Belfast, UK). Fluorescence data were collected and processed by NIS-elements (Nikon, Tokyo, Japan) and Photoshop CS6. Fluorescence intensities were measured by ImageJ 1.52k. Data were analyzed by Excel for Mac 2016.
6
Graphitized Carbon Nano-Electrodes Fabrication
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All the materials and chemicals used in this work were of analytical grade and used as received. Mesoporous graphitized carbon nanopowder (MGC, >99.95%, <500 nm DLS, 50–100 m2/g, <200 nm particle size), polyvinylpyrrolidone (PVP, Mw~1,300,000), ethanol anhydrous (EtOH 99,8%), polyethyleneimine (PEI, Mw~423), acetic acid (HAc, ≥99%), formic acid (FA, ≥98%), methanol (MeOH, ≥99.8%), acetone (Ac, ≥99.5%), triethylamine (TEA, ≥99.5%), and butylamine (ButA, ≥99.5%) were purchased from Merck KGaA (Darmstadt, Germany).
Interdigitated electrodes (IDEs), supplied by Micrux Technologies (Gijón, Spain), were fabricated on glass substrates (IDE dimensions: 10 × 6 × 0.75 mm, Pt/Ti electrodes, 120 pairs, 10 μm wide × 5 mm long × 150 nm thick, with a 10 μm gap). Prior to use, the electrodes were cleaned with a soap solution and a “base piranha” mixture at 60 °C for approximately 30 min (3:1, v:v, ammonia water, and hydrogen peroxide water solution), followed by rinsing with Milli-Q water (~18 MΩ cm).
Interdigitated electrodes (IDEs), supplied by Micrux Technologies (Gijón, Spain), were fabricated on glass substrates (IDE dimensions: 10 × 6 × 0.75 mm, Pt/Ti electrodes, 120 pairs, 10 μm wide × 5 mm long × 150 nm thick, with a 10 μm gap). Prior to use, the electrodes were cleaned with a soap solution and a “base piranha” mixture at 60 °C for approximately 30 min (3:1, v:v, ammonia water, and hydrogen peroxide water solution), followed by rinsing with Milli-Q water (~18 MΩ cm).
7
Adsorption of Chromium from Aqueous Solution
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Polyethyleneimine (PEI), sodium hydroxide (NaOH), sodium hypochlorite (NaClO), and a chromium standard solution (1000 mg L−1) were purchased from Merck Co., Inc. (Darmstadt, Germany). The cellulose fibers (Cel) were derived from bamboo waste. All chemicals were of analytical grade and were used without purification.
A Thermo Scientific spectrometer (Thermo Nexus 470FT-IR, Nicorette, Waltham, MA, USA) was used to obtain the Fourier transform infrared (FT-IR) spectra of the different materials in the range 400–4000 cm−1, while a scanning electron microscope (Hitachi FE-SEMS-4800, Hitachi City, Japan) was used to determine their surface morphologies. The phase purity and crystal structure were examined using an X-ray diffractometer (Bruker/D8 Advance, Billerica, MA, USA). Thermogravimetric analyses (TGA) were performed using a Thermo Scientific SDT Q600 series Thermogravimetric Analyzer (TA instrument, New Castle, DL, USA). The concentration of chromium was determined using an atomic absorption spectrophotometer (PerkinElmer PinAAcle 900, Waltham, MA, USA).
A Thermo Scientific spectrometer (Thermo Nexus 470FT-IR, Nicorette, Waltham, MA, USA) was used to obtain the Fourier transform infrared (FT-IR) spectra of the different materials in the range 400–4000 cm−1, while a scanning electron microscope (Hitachi FE-SEMS-4800, Hitachi City, Japan) was used to determine their surface morphologies. The phase purity and crystal structure were examined using an X-ray diffractometer (Bruker/D8 Advance, Billerica, MA, USA). Thermogravimetric analyses (TGA) were performed using a Thermo Scientific SDT Q600 series Thermogravimetric Analyzer (TA instrument, New Castle, DL, USA). The concentration of chromium was determined using an atomic absorption spectrophotometer (PerkinElmer PinAAcle 900, Waltham, MA, USA).
8
Rat Neuron Culture and MeHg Exposure
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Primary cultures of rat cerebrocortical and hippocampal neurons were established using a modified version of the protocol described by Unoki et al. (2012) and Fujimura and Usuki (2015) . In brief, the cerebral cortex and hippocampus were dissected from 20-day-old Sprague-Dawley rat embryos (Clear Japan, Tokyo, Japan). The tissues were digested with papain (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 37°C for 30 min and dissociated by gentle pipetting. The suspensions were filtered and centrifuged at 110 × g at room temperature for 5 min, and the cells were cultured in polyethyleneimine (Merck, Darmstadt, Germany)-coated wells in tissue-culture plates at a density of 4 × 10 4 cells/cm 2 in neurobasal medium supplemented with B27 and 2 mM GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA). Culture in this serum-free medium reportedly suppresses the growth of glial cells by more than 99.5%. Seven days after plating, the cells were exposed to MeHg chloride (Tokyo Chemical Industry, Tokyo, Japan). MeHg chloride was dissolved in dimethyl sulfoxide (DMSO; Nacalai Tesque, Kyoto, Japan) and added to the medium at a DMSO concentration of 0.1% (Fujimura and Usuki, 2018) .
All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institute for Minamata Disease.
All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institute for Minamata Disease.
9
Zebrafish Hepatic Cell Isolation
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The hepatic zebrafish samples were collected and placed into Ca2+/Mg2+-free HBSS Medium (Gibco) for rinsing three times. After the samples were chopped, they were digested in collagenase (VETEC, Type II) at 28°C for 30 min. The cell suspensions were filtered through 0.45-μm filters. Cells were then collected by centrifugation. Counted cells were seeded into plates with Polyethyleneimine (ALDRICH), and cultivated in DMEM/F-12(HAM)1:1 (BI, Biological Industries) with 10% penicillin-streptomycin solution (Biosharp) for 24 h at 28°C in humidified atmosphere with 5% CO2. After 24 h of incubation, cells were used for the glucose uptake assay (28 (link)).
10
Carrageenan-Alginate Chitin Nanocomposite
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k-Carrageenan (Mr: 154,000; sulfate ester ~25 %), alginic acid sodium salt from brown algae (CAS # 9005-38-3), Chitin from crab shells and p-nitrophenyl-β-d -N-acetyl glucoseaminide (PNP-β-GlcNAc) were purchased from Fluka Biochemika Co. (USA). Polyethyleneimine (MW: 423), Cat # 468533, was obtained from Aldrich. All other reagents were of the purest grade commercially available.
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