Adapted from Rodrigues et al (20 ). Assays were carried out in modified Sauton’s media (m-Sauton’s) with 0.05% Tween-80 (102 ), without pantothenic acid or L-leucine supplementation, from which ferric ammonium citrate and zinc sulfate were omitted to avoid optical interference and for convenience respectively. Mid-logarithmic cultures between OD600 0.4-0.8 were harvested in 50mL Falcon tubes by centrifugation for 9 minutes at 3220 x g, washed and resuspended at OD600 0.4-0.8 in m-Sauton’s, then aliquoted into a 96-well black optical-bottom plate (Costar) in triplicate with drug or vehicle as appropriate. Ethidium bromide (EtBr) (Fisher Scientific, 10132863) in m-Sauton’s was added to each well to a final concentration of 1μg/mL (25% of EtBr’s reported MIC for H37Rv (103 ). Intrabacterial accumulation of EtBr was measured in situ using a plate reader (CLARIOstar, BMG LabTech) by fluorimetry at 530±25 nm excitation and 590±20 nm emission every 60-120 seconds. Measurements were continued until steady state was reached, typically after 90 minutes. For ease of comparison between drugs and experiments, the mean final 10 values measured in steady state under any one condition were expressed as a percentage of the mean final 10 values measured in steady state without drug present.
Ethidium bromide etbr
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ethidium bromide (EtBr) is a fluorescent dye used to detect and visualize DNA and RNA in molecular biology applications. It intercalates between the base pairs of nucleic acids, enabling the visualization of DNA and RNA fragments under ultraviolet (UV) light.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dapi 4 6 diamidino 2 phenylindole dihydrochloride acridine orange, by Thermo Fisher Scientific (3 mentions)
Clariostar, by BMG Labtech (2 mentions)
96 well black optical bottom plate, by Corning (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Bcl 2, by Santa Cruz Biotechnology (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Translucent pet membrane, by BD (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Diethyl ether, by Avantor (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Deuterated chloroform cdcl3, by Eurisotop (1 mentions)
Silica gel 60, by Carl Roth (1 mentions)
L α phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Propidium iodide staining solution, by BD (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
9 protocols using ethidium bromide etbr
1
Assaying Intrabacterial Drug Accumulation
2
Cystamine-Bisacrylamide Mediated siRNA Delivery
3
Assaying Intrabacterial Drug Accumulation
4
Cationic Liposomal Nanoparticle Formulation
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Freeze-dried biomass of Sulfolobus acidocaldarius was obtained from SiT (Surface & Interface Technologies, Rosenhof GmbH, Heiligenstadt, Germany). N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) was a gift from Lipoid (Ludwigshafen, Germany). L-α-Phosphatidylcholine was purchased from Avanti Polar Lipids (Alabama, USA) and cholesterol was obtained from Sigma-Aldrich (Taufkirchen, Germany). DNA intercalating dyes gel-red (10.000x in DMSO) and ethidium bromide (EtBr) were obtained from Invitrogen (California, USA) and Sigma-Aldrich (Missouri, USA), respectively. Purified plasmid pCMV-luc (pDNA) was purchased from Plasmid Factory (Bielefeld, Germany). Silica gel 60 (400–230 mesh) was obtained from Carl Roth GmbH (Karlsruhe, Germany). Reversed phase Chromabond C-18 and HPTLC (high performance thin layer chromatography) plates were bought from Macherey-Nagel (Weilmünster, Germany). Organic solvents chloroform (CHCl3), methanol (MeOH), and diethyl ether (DE) were obtained from VWR International (Pennsylvania, USA). Hydrochloric acid was purchased from Sigma-Aldrich. For 1H-NMR, deuterated chloroform (CDCl3) was obtained from Euriso-Top (Gif-sur-Yvette Cedex, France). All solvents used were of HPLC grade.
5
Chitosan-PEI Nanoparticle Synthesis Protocol
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Low-molecular-weight CHI, with a viscosity of 20–300 cP, Mw 50–190 kDa, and 75%–85% deacetylation, was obtained from Aldrich (St Louis, MO, USA). PEI with an Mw of 1,800 Da (PEI1800) and poly(ethylene glycol) diglycidyl ether (EX-810) with an Mw of 512 Da were also purchased from Aldrich. PEI with an Mw of 600 Da (PEI600) was purchased from Alfa Aesar (Ward Hill, MA, USA). Dimethylsulfoxide (DMSO) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were supplied by Sigma (St Louis, MO, USA). The 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), fluorescein isothiocyanate (FITC), and ethidium bromide (EtBr) were provided by Invitrogen (Camarillo, CA, USA). The propidium iodide (PI) staining solution was supplied by BD Bioscience (San Jose, CA, USA). The Dulbecco’s modified eagle medium (DMEM), antibiotic-antimycotic solution, and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Plasmid-encoding enhanced green fluorescence protein (GFP) (pEGFP-N1) was purchased from Clontech Laboratories, Inc., (Palo Alto, CA, USA). Plasmid-encoding GDNF and GFP (pAAC-MCS-rGDNF-IRES-hrGFP, abbreviated as pGDNF-GFP) was donated by Dr Wu at the Materials Engineering Department of Tatung University, Taiwan.
6
Tetrachloroauric Acid Cytotoxicity Assay
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Tetrachloroauric acid (HAuCl4), TA, propidium iodide (PI), caspase-3 and caspase-9 activity assay kit were bought from Sigma Aldrich (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Annexin-V/FITC kits and rhodamine 123 were obtained from Calbiochem. All cell lines were bought from National Centre For Cell Science (NCCS), Pune, Govt. of India. Fetal bovine serum, Dulbecco's Modified Eagle Medium, Pen-strep (penicillin and streptomycin) antibiotic, and trypsin were obtained from Gibco BRL (Grand Island, USA). Cell culture plastic wares were obtained from NUNC (Denmark) and protein assay reagent, BCA, was purchased from Pierce. DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), acridine orange (AO), and ethidium bromide (EtBr) were supplied by Invitrogen (California). All primary and secondary antibodies were purchased from Cell Signaling Technology (CST). All other reagents were bought from Sigma-Aldrich Company, USA.
7
MTT, DCFH-DA, and DPPH Protocols for Cellular Assays
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3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), 2', 7'-dichlorodihydrofluorescin diacetate (DCFH-DA), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and deoxyribose were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, and 0.05% trypsin-0.53 mM EDTA were purchased from Grand Island Biological Company (Grand Island, NY, USA). TRIzol reagent, oligodT18 primer, murine maloney leukemia virus (MMLV) reverse transcriptase, RNase inhibitor, reverse transcriptase buffer, dNTPs, ethidium bromide (EtBr), and agarose were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Chemicals used were of standard analytical grade.
8
MTT Assay for Cytotoxicity Evaluation
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is the product of SRL, N-acetyl cysteine (NAC) was purchased from Sigma Aldrich, and HCT116 and HEK293 cell lines were obtained from the National Centre For Cell Science (NCCS), Pune, Govt. of India. HCT 116 cell lines were cultured in DMEM Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin antibiotics, where trypsin and ethylenediaminetetraacetic acid (EDTA) were all purchased from Gibco BRL. Tissue culture plastic wares were obtained from NUNC (Roskidle, Denmark). DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), acridine orange (AO), and ethidium bromide (EtBr) were obtained from Invitrogen (California) and BCA protein assay reagent from Fermentus (EU). The primary antibodies for Bcl2, Bax, caspase 3, cleaved caspase-3, caspase-9, cleaved caspase-9, cytochrome c, P53, cleaved PARP, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). DCF-DA was obtained from Sigma-Aldrich (MO, USA). An Annexin-V FITC apoptosis detection kit was obtained from Calbiochem (CA, USA). Doxorubicin was purchased from Sigma Aldrich.
9
Apoptosis Signaling Pathway Analysis
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MTT and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA); p-NBT-BCIP systems were from Amresco (Solon, OH, USA); RPMI 1640, FBS (Fetal Bovine Serum), and penicillin—streptomycin—neomycin were from Gibco-BRL (Grand Island, NY, USA); tissue-culture plasticware was from Nunc (Roskilde, Denmark); Bradford protein assay reagent was from Fermentas (Pittsburgh, PA, USA); DAPI (4',6-diamidino-2-phenylindole dihydrochloride), acridine orange (AO), and ethidium bromide (EtBr) were from Invitrogen (Carlsbad, CA, USA); rabbit and goat anti-BAX, BAD, BCL-2, p38, p-P38, ERK, p-ERK, JNK polyclonal and secondary antibodies in alkaline phosphatase, and FITC and PE-conjugated were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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