Surface labeling was done with Sulfo-NHS-SS-Biotin, which purchased from Thermo Fisher (Waltham, MA, USA). Synthetic oligonucleotides for site-specific mutagenesis were from IDT (Coraiveille, IA, USA). For pHi measurements, 2′,7-bis(2-carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). For transfection, the LipofectamineTM 2000 reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). For protein stability studies, cycloheximide was purchased from Santa Cruz Biotechnology Inc, (Dallas, TX, USA). Fisher Scientific, BDH (Toronto, ON, Canada) or Sigma-Aldrich (St. Louis, MO, USA) supplied other chemicals of analytical grade. The plasmid pYN4+ has an HA (hemagglutinin) tag, as described earlier [25 (link)], and was used to express cDNA for the human NHE1 protein. The plasmid pmEmerald-dectin-1A-N10 was a generous gift from Dr. N. Touret, of the Department of Biochemistry, University of Alberta. The polyclonal anti-GFP antibody was a generous gift of Dr. Luc Berthiaume, Department of Cell Biology, University of Alberta.
2 7 bis 2 carboxyethyl 5 6 carboxyfluorescein acetoxymethyl ester bcecf am
Manufactured by Thermo Fisher Scientific
Sourced in United States
2',7-bis(2-carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM) is a fluorescent dye compound used for the measurement of intracellular pH in living cells. It is a cell-permeant indicator that becomes fluorescent upon hydrolysis by intracellular esterases.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (3 mentions)
Sulfo nhs ss biotin, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Santa Cruz Biotechnology (1 mentions)
Streptavidin agarose, by Thermo Fisher Scientific (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Trypan blue solution, by Merck Group (1 mentions)
Tissue culture reagents, by Thermo Fisher Scientific (1 mentions)
Paeoniflorin, by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rabbit anti nrf2, by Santa Cruz Biotechnology (1 mentions)
Mouse anti icam 1, by Santa Cruz Biotechnology (1 mentions)
Daf fm diacetate, by Thermo Fisher Scientific (1 mentions)
Goat anti vcam 1, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg, by Enzo Life Sciences (1 mentions)
Mouse anti p iκb α, by Santa Cruz Biotechnology (1 mentions)
Mouse anti nf κb, by Santa Cruz Biotechnology (1 mentions)
7 protocols using 2 7 bis 2 carboxyethyl 5 6 carboxyfluorescein acetoxymethyl ester bcecf am
1
Molecular Labeling and Transfection Techniques
2
Fluorescent Protein Labeling Protocol
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2', 7-bis (2-carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM) was from Molecular Probes, Inc. (Eugene, OR). Sulfo-NHS-SS-biotin was purchased from Pierce and synthetic DNA was purchased from IDT (Coraiveille, IA, USA). Streptavidin agarose was obtained from Thermo Scientific. Anti HA (hemagglutinin) tag primary antibody was from Pierce. GAM-HRP was obtained from Jackson ImmunoReseach Lab Inc(Bio/Can, Mississauga, Canada). Other chemicals that were used were of analytical grade and were purchased from Fisher Scientific (Ottawa, ON, Canada), Sigma (St. Louis, MO, USA) or BDH (Toronto, ON, Canada). The plasmid pYN4+ contains the human NHE1 protein with a HA tag and has been described earlier [11 (link)].
3
Molecular Tools for NHE1 Protein Analysis
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Synthetic oligonucleotides were obtained from IDT (Coralville, IA, USA). Sulfo-NHS-SS-biotin was bought from Thermo Fisher (Waltham, MA, USA). 2′,7-Bis(2-carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM) was obtained from Molecular Probes, Inc. (Eugene, OR, USA). LipofectamineTM 2000 reagent was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Toronto, ON, Canada) supplied the other analytical-grade chemicals used in this study. The plasmid pYN4+ was used to express cDNA for the full-length human NHE1 protein; it has a triple HA (hemagglutinin) tag, is completely functional, and has been described previously [59 (link)]. Anti-NHE1 protein antibody (#611774) was purchased from BD Biosciences. Anti-tubulin protein antibody was purchased from Sigma (St. Louis, MO, USA). The plasmid pSpCas9(BB)-2APuro (pX459) V 2.0 was a generous gift from Dr. M. Michalak, Department of Biochemistry, University of Alberta. DU 145 cells and HT1080 cells were obtained from ATCC—catalog numbers HTB-81 and CCL-121, respectively.
4
Synthetic Oligonucleotides for Site-Specific Mutagenesis
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Synthetic oligonucleotides for site-specific mutagenesis were obtained from IDT (Coraiveille, IA, USA). Sulfo-NHS-SS-biotin was purchased from Thermo Fisher (Waltham, MA, USA). 2′,7-bis(2-carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes, Inc. (Eugene, OR, USA). LipofectamineTM 2000 reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Sigma-Aldrich (St. Louis, MI, USA) or Fisher Scientific, (Toronto, ON, Canada) supplied other chemicals of analytical grade. The plasmid pYN4+ has a hemagglutinin (HA) tag, is described earlier [43 (link)], and was used to express cDNA for the full-length human NHE1 protein. The Ni-NTA resin was obtained from QIAGEN (Toronto, ON, Canada).
5
Intracellular pH Regulation Assay
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All general laboratory chemicals and Trypan Blue solution (Catalogue No. T8154) were purchased from Sigma-Aldrich (Budapest, Hungary). We obtained 2,7-bis-(2-carboxyethyl)-5 (6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) from Invitrogen (Watham, MA, USA), prepared (2 µmol/L) in dimethyl sulfoxide and stored at −20 °C. Nigericin (10 µM) was dissolved in ethanol and stored at −20 °C. High-Capacity Reverse Transcription Kit, TaqMan gene expression Master Mix, lipofectamine 2000 transfection reagent were purchased from Thermo Fisher Scientifc (Watham, MA, USA). Cell Counting Kit-8 was from Dojindo Molecular Technologies (Rockville, MD, USA). Citotoxicity detection kit (LDH) was obtained from Roche (Catalogue No. 11644793001, Roche). SLC9A1 siRNAs (siRNA ID: 119643) was from Life Technologies. The standard Na+-HEPES solution contained (in mM): 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 D-glucose and 10 Na-HEPES. NH4Cl-HEPES solution was supplemented with 20 mM NH4Cl, while NaCl concentration was lowered to 110 mM. HEPES-buffered solutions were gassed with 100% O2, and their pH was set to 7.5 with HCl.
6
Phytochemical Analysis and Anti-inflammatory Effects
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Ferulic acid and 5-hydroxymethyl-2-furaldehyde (5-HMF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Albiflorin and paeoniflorin were the products of Wako (Osaka, Japan). Nodakenin was purchased from NPC BioTechnology Inc. (Daejeon, Korea). The purity of all reference standards was ≥98.0%. HPLC-grade methanol, acetonitrile, and water were obtained from J.T.Baker (Phillipsburg, NJ, USA). Glacial acetic acid, analytical reagent grade, was purchased from Junsei (Tokyo, Japan). RPMI 1640, fetal bovine serum, TNF-α, tissue culture reagents, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy-methylester (BCECF-AM), DAF-FM diacetate, and CM-H2DCFDA, Alexa Fluor 488 and 594 conjugated second antibodies were purchased from Invitrogen (San Diego, CA). Biotin 3′ End DNA Labeling Kit, LightShift® Chemiluminescent EMSA Kit, Biodyne® Precut Nylon Membranes, Lipofectamine LTX reagent, and Renilla-Firefly Luciferase Dual Assay Kit were purchased from Pierce Biotechnology (Rockford, USA). Primary antibodies, including mouse anti-ICAM-1, goat anti-VCAM-1, rabbit anti-E-selectin, mouse anti-NF-κB, mouse anti-p-IκB-α, rabbit anti-HO-1, and rabbit anti-Nrf2, were purchased from Santa Cruz Biotechnology (CA, USA). Donkey anti-goat IgG-H+I were purchased from Bethyl (Montgomery, USA) and goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Enzo (Farmingdale, USA).
7
Monitoring Intracellular pH Regulation
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Prostaglandin E2 (PGE2) and forskolin were purchased from Sigma. pH-sensitive fluorescent dye, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein acetoxymethyl ester (BCECF-AM), was from Invitrogen. Anti-CFTR, anti-SLC26A6, anti-P38, anti-phospho-P38, and anti-β-actin antibodies were from Santa Cruz. All other chemicals in solutions were obtained from Sigma and Calbiochem.
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