Quercetin, protoporphyrin IX (PpIX), dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, USA. Ethanol, superoxide dismutase (SOD), catalase, ethidium bromide, hypoxanthine, diethylenetriaminepentaacetic Acid (DTPA), trifluoroacetic acid, acetonitrile, iron(II) perchlorate, hydrogen peroxide, fluorescein, phosphoric acid, Dulbecco’s phosphate-buffered saline (D-PBS) were purchased from FUJIFILM Wako Pure Chemical Corporation., Japan. 3’-(p-aminophenyl) fluorescein (APF) was purchased from Sekisui Medical Co. Ltd., Japan. Dihydroethidium DHE was purchased from Invitrogen, CA. Compound libraries (Core library; for pilot screening) were provided by drug discovery initiative (DDI), the University of Tokyo (https://www.ddi.u-tokyo.ac.jp/en/ ). WST-8 was purchased from Dojin Laboratoies, Japan. Xanthine oxidase was purchased from Calbiochem (USA).
Ethidium bromide
Manufactured by Fujifilm
Sourced in Japan
Ethidium bromide is a fluorescent dye commonly used in molecular biology laboratories for the detection and visualization of nucleic acids, such as DNA and RNA, during gel electrophoresis. It intercalates between the base pairs of nucleic acids, emitting an orange-red fluorescence when exposed to ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Rtaq polymerase, by Toyobo (2 mentions)
Phosphoric acid, by Fujifilm (1 mentions)
Hydrogen peroxide, by Fujifilm (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
Ethanol, by Fujifilm (1 mentions)
Quercetin, by Merck Group (1 mentions)
Acetonitrile, by Fujifilm (1 mentions)
Catalase, by Fujifilm (1 mentions)
Trifluoroacetic acid, by Fujifilm (1 mentions)
Hypoxanthine, by Fujifilm (1 mentions)
Superoxide dismutase sod, by Fujifilm (1 mentions)
Diethylenetriaminepentaacetic acid, by Fujifilm (1 mentions)
Dulbecco s phosphate buffered saline d pbs, by Fujifilm (1 mentions)
3 p aminophenyl fluorescein apf, by Sekisui (1 mentions)
Acridine orange, by Dojindo Laboratories (1 mentions)
Generuler 100 bp plus dna ladder, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Nippon Gene (1 mentions)
Nucleospin rna kit, by Takara Bio (1 mentions)
Takara ex taq, by Takara Bio (1 mentions)
Gentra puregene cell kit, by Qiagen (1 mentions)
10 protocols using ethidium bromide
1
Optimizing Oxidative Stress Assays
2
Radiation-Induced Apoptosis and Necrosis Assay
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Radiation-induced apoptosis and necrosis were determined by fluorescence staining with acridine orange (Dojindo laboratories, Kumamoto, Japan) and ethidium bromide (Wako, Osaka, Japan) (Supplementary Fig. 1 ) as previously described [19 (link), 20 (link)]. The percentages of apoptotic or necrotic cells were calculated with normalization to the total number of cells. The dose–response curves were generated using the polynomial approximation by KaleidaGraph software (Hulinks, Tokyo, Japan) (Supplementary Fig. 2 ) as previously described [21 (link)]. The RBE values were calculated using the coefficients of the linear components of the curve-fitting–formulae (Supplementary Table 1 ). At least 400 cells were counted per sample.
3
Reverse Transcription and PCR Amplification of DGK Isoforms
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The isolation of total RNA, reverse transcription and PCR amplification were performed as previously described [26] (link). The PCR amplification was performed using rTaq polymerase (TOYOBO) and the following mouse DGKα~θ-specific oligonucleotide primers. The DGKα primers were the following: forward primer (nucleotide positions 333–352, 5′-GATGGCCAAAGAGAAGGGCC-3′) and reverse primer (nucleotide positions 658–675, 5′-GTCTTCTGGCCGGCCACC-3′). The PCR conditions were as follows: 94 °C for 3 min, 32 cycles of 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 0.5 min, and 72 °C for 15 min. The DGKβ primers were the following: forward primer (nucleotide positions 435–457, 5′-CCATGACAAACCAGGAAAAATGG-3′) and reverse primer (nucleotide positions 847–866, 5′-CCTCGGGTCTTCCTCTTTCG-3′). The DGKγ primers were the following: forward primer (nucleotide positions 458–477, 5′-GATGAGCGAAGAACAATGGG-3′) and reverse primer (nucleotide positions 965–981, 5′-CCTGAGGTCGCCCGGTC-3′). The DGKδ [27] (link), DGKκ [28] (link), DGKη [28] (link), DGKε [29] (link), DGKζ [29] (link), DGKι [29] (link) and DGKθ [29] (link) primers were previously described. The amplified PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide (Wako Pure Chemical, Osaka, Japan). A mouse whole brain was used for positive control.
4
PCR Product Molecular Weight Analysis
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After amplification, 5 μL of PCR product was subjected to analysis on 2% agarose gel (Bioline Reagents Ltd., London, UK) in 0.5X TBE buffer (Omega BioTek, Inc; Norcross, Georgia) to determine the molecular weight of the amplified DNA fragment. The 5 μL GeneRuler 100 bp Plus DNA ladder (Thermo Scientific; Vilnius, Lithuania) was loaded onto the same agarose gel as a molecular weight standard. Subsequently, the gel was stained with ethidium bromide (Wako, Wako Pure Chemical Industries, Ltd.; Tokyo, Japan) and destained by soaking it in water. Electrophoresis was performed on horizontal electrophoresis equipment (Mupid-Exu; Chuo-ku, Japan) for 30 min at a constant 100 Volts. Subsequently, the gel was visualized using a UV Transilluminator (SynGene; Cambridge, UK), enabling a comparison between the migration patterns of the DNA ladder bands and the PCR products.
5
Gene Expression Analysis by RT-PCR
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Total RNA isolation, reverse transcription and PCR amplification were performed as previously described [22 (link)]. PCR amplification was performed using rTaq polymerase (Toyobo, Osaka, Japan) and the following mouse Prl-, Gh- and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific oligonucleotide primers in the outlined conditions: Prl, forward primer (5′-ATGACCATGAACAGCCAGGGGT-3′) and reverse primer (5′-TTCCTCAATCTCTTTGGCTCTTGATAGGAT-3′) with PCR conditions of 94 °C for 3 min, then 30 (DGKη-KO) or 35 (WT) cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, and finally 72 °C for 5 min; Gh, forward primer (5′-ATGGCTACAGACTCTCGGACCTC-3′) and reverse primer (5′-CTGCATCAGAGCCTGGATGCC-3′) with PCR conditions of 94 °C for 3 min, then 34 (DGKη-KO) or 39 (WT) cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min, and finally 72 °C for 5 min; GAPDH, forward primer (5′-TCGGTGTGAACGGATTTGGCCGTATT-3′) and reverse primer (5′-CATGTAGGCCATGAGGTCCACCAC-3′) with PCR conditions of 94 °C for 3 min, then 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min; and finally 72 °C for 5 min. The amplified PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide (Wako Pure Chemical Industries, Osaka, Japan).
6
RT-PCR Analysis of Immune Cell Markers
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Total RNA was extracted from 5.0 × 10 5 sorted cells of each cell population using a NucleoSpin® RNA kit (TaKaRa, Japan) and reverse transcribed into cDNA using M-MLV Reverse Transcriptase (NIPPON GENE) with Oligo d(T)16 primers. Specific primer sets for CD4-1, CD8α, TCRβ, TCRγ, Lck, and EF1-α were used for RT-PCR, as shown in Table 1 (Barreda et al., 2005; (link)Matsuura et al., 2014; (link)Miyazawa et al., 2018; (link)Nonaka et al., 2008; (link)Toda et al., 2011b Toda et al., , 2011a;; Accession.No, NC_039269.1.) . The PCR conditions were as follows: one cycle of 95°C for 2 min, 30-37 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 30 s. The PCR reactions were performed in 24.5-μl reaction mixtures containing 10× Ex Taq Buffer, dNTP Mixture (2.5 mM each), 1.0 µM of each primer, TaKaRa Ex Taq (5 units/μl) (TaKaRa, Japan), and 0.5 μl of cDNA template. The PCR products were electrophoresed in 1.3% agarose gels and visualized by staining the gel with 0.5 µg/ml ethidium bromide (Wako, Japan). Images of the PCR products were photographed using Gel Scene Tablet (ASTEC, Japan).
7
PCR Detection of Resistance Genes
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Genomic DNA of drug-resistant cells was extracted using a Gentra Puregene Cell Kit (Qiagen; Venlo, NL) following the manufacturer’s instructions, and PCR was performed with KOD One PCR Master Mix following the manufacturer’s instructions. The following PCR primers were used to detect each resistant gene on the HAC/MAC: bsd F (5ʹ-CAACAGCATCCCCATCTCTG-3ʹ) and #21cen6R (5ʹ-CCCGGCCAGATTCAGATTTTTATTAGGG-3ʹ) with a product of 3,641 bps for the detection of the blasticidin S-resistant gene on 21HAC22 (link) (Figures S1 A, S11 A, and S11B), kj_neo (5ʹ-CATCGCCTTCTATCGCCTTCTTGACG-3ʹ) and m11_7R (5ʹ-CACTCTTTACCCCTCACCGCTAACCTTG-3ʹ) with a product of 6,630 bps for the detection of the neomycin resistant-gene on MAC127 (link) (Figures S1 B, S11 C, and S11D), and EF1a Fw (5ʹ-CACTGAGTGGGTGGAGACTGAAGTTAGG-3ʹ) and Neo817 (5ʹ-GCAGCCGATTGTCTGTTGTG-3ʹ) with a product of 370 bps for the detection of the neomycin resistant-gene on modified 21HAC26 (link) (Figures S1 D and S11 E). The PCR products were then resolved by electrophoresis on Agarose S gels (Fujifilm Wako) followed by staining with ethidium bromide (Fujifilm Wako). FastGene 1 kb DNA Ladder Plus (Nippon Genetics Co.; Tokyo, Japan) was used as a band size marker.
8
RT-PCR Analysis of IL-6 Expression
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Total RNA was extracted from cells with ISOGEN reagent (Nippon Gene Co. Ltd., Tokyo, Japan). The first-strand cDNA was synthesized from total RNA with PrimeScript Reverse Transcriptase (Takara Bio, Shiga, Japan). GAPDH mRNA was determined as a positive control. PCR was done by incubating cDNA with appropriate primers (0.5 μM each), Blend Taq polymerase (1.25 U: Toyobo, Osaka, Japan) and deoxynucleotide mix (0.2 mM each: Toyobo). The reaction conditions were as follows: 94°C for 2 min, followed by 30 cycles of 94°C for 30 sec, 56°C for 1 min, and 72°C for 2 min, with final extension at 72°C for 10 min. The primer sequences were as follows: IL-6 (236 bp), 5’-agagtagtgaggaacaagcc-3’ (sense) and 5’-tacatttgccgaagagccct-3’ (anti-sense); GAPDH (258 bp), 5’-agaaggctggggctcatttg-3’ (sense) and 5’-aggggccatccacagtcttc-3’ (anti-sense). The products were then subjected to 2% agarose gel electrophoresis. Bands were stained with ethidium bromide (Wako) and photographed.
9
In Vitro RNA Cleavage Assay of HBV Epsilon
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The in vitro RNA cleavage assay analysis was performed as described previously17 (link). The reaction mixture containing 7.5 µM 5′-FAM conjugated HBV epsilon RNA (Table S1 ) and 2 µM recombinant MCPIP1 (ORIGENE, TP301381) was incubated at 37 °C for 4 h in 25 mM tris–HCl at pH 7.9, 150 mM NaCl, 10% Glycerol, 2.5 mM MgCl2, 1 mM DTT, 0.5 mM EDTA, and 0.05 mM ZnCl2. The reaction was terminated by freezing in liquid nitrogen. The samples were then mixed with 2 × RNA Loading Buffer with ethidium bromide (Wako, 185-02561), denatured at 70 °C 10 min, chilled on ice, and loaded on 15% SuperSepTMRNA (Wako, 194-15881). Fluorescence was detected using LAS 4000.
10
Quinolone Antimicrobial Inhibition Assay
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WQ-3810 was kindly provided by Wakunaga Pharmaceutical Co., Ltd. The inhibitory effect of WQ-3810 was assayed by comparing it with that of ciprofloxacin and nalidixic acid, which were tested in a previous study [25] . ciprofloxacin is clinically crucial for treating infectious human diseases as explained in the introduction [8] . On the other hand, nalidixic acid is less important as a therapeutic agent because other more effective quinolones are available, but it is important as a standard for screening quinolone-resistant bacteria [28] . The chemical structures of these quinolones are shown in Figure 1. ciprofloxacin and nalidixic acid were purchased from LKT Laboratories, Inc. (St Paul, MN, USA) and Wako Pure Chemical Industries, Ltd (Tokyo, Japan), respectively. Relaxed pBR322 DNA was purchased from John Innes Enterprises Ltd (Norwich, UK) and Ethidium bromide was obtained from Wako Pure Chemical Industries, Ltd.
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