Live cell imaging and calcium mobilization experiments were performed as described previously [1 (link),19 (link)]. Cells were preincubated with ARL 67156 trisodium salt (ARL) (Sigma-Aldrich (St. Louis, MO, USA)) (10, 20, 50 nM) [25 (link),26 (link)], Fluo-4AM (Thermo Fisher, Waltham, MA, USA) (1:100) and the counterstain SirActin (Cytoskeleton, Inc., Denver, CO, USA) (1:1000) for 20 min at 37 °C. Images were collected every 3 s for 45 min on the Zeiss Axiovert LSM 880 confocal microscope (Zeiss, Thornwood, NY, USA) utilizing the 20× air objective. Scratch wounds or agonist stimulation by ATP, 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP) (Sigma-Aldrich (St. Louis, MO, USA)), or ATP (Sigma-Aldrich (St. Louis, MO, USA)) were performed after 100 frames (5 min). All injuries were made using a 25-gauge needle.
2 3 o 4 benzoylbenzoyl atp bzatp
Manufactured by Merck Group
Sourced in United States
2,3-O-(4-benzoylbenzoyl)-ATP (BzATP) is a laboratory reagent that functions as a synthetic analog of adenosine triphosphate (ATP). It is used as a tool to investigate the properties and behavior of ATP and related biological processes in research settings.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine triphosphate (atp), by Merck Group (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (2 mentions)
Axiovert lsm 880 confocal microscope, by Zeiss (1 mentions)
Sir actin, by Cytoskeleton (1 mentions)
Phosphate colorimetric assay kit, by Abcam (1 mentions)
Fluo 3 acetoxymethyl ester fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Az11645373, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Ar c 118925xx, by Bio-Techne (1 mentions)
Cellmask deep red plasma membrane stain, by Thermo Fisher Scientific (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
A438079 hydrochloride, by Bio-Techne (1 mentions)
Fluo 3am fluorescent dye, by Thermo Fisher Scientific (1 mentions)
10panx inhibitory peptide, by Bio-Techne (1 mentions)
Yo pro 1 dye, by Merck Group (1 mentions)
Oxatp, by Merck Group (1 mentions)
Pherastar fs plate reader, by BMG Labtech (1 mentions)
4 protocols using 2 3 o 4 benzoylbenzoyl atp bzatp
1
Live Cell Calcium Imaging of ATP Signaling
2
Physiological Salt Solution Preparation
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The physiological salt solution contained (mmol/L) 140 NaCl, 5.4 KCl, 1 CaCl2, 1 Na2HPO4, 0.5 MgCl2, 5 glucose, and 5 HEPES (pH = 7.4). Thapsigargin (Tg) was procured from Calbiochem (San Diego, CA). 2-Aminoethyldiphenyl borate (2APB), ethidium bromide, AZ11645373, and 2′,3′-O-(4-benzoyl) benzoyl ATP (BzATP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Fluo-3 acetoxymethylester (Fluo-3 AM) was from Molecular Probes (Eugene, OR), lymphocyte separation medium LSM-1077 was from PAA Laboratories GmbH (Pasching, Austria), and fetal bovine serum (FBS) and RPMI-1640 medium were from GIBCO (Grand Island, NY, USA). PE-conjugated anti-CD14, FITC-conjugated IgG2a, and anti-P2X7 (extracellular) FITC were from Sigma-Aldrich (St. Louis, MO, USA). The phosphate colorimetric assay kit was from BioVision (Hayward, CA, USA). All other chemicals were purchased from Sigma-Aldrich.
3
Molecular Probes for Cell Signaling
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ATP, 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP), and UTP were all purchased from Sigma-Aldrich (St. Louis, MO). A luciferase-based ATP Determination Kit was purchased from Invitrogen (Carlsbad, CA). A438079 hydrochloride, AR-C 118925XX, 10Panx Inhibitory peptide, and scramble control peptide were purchased from Tocris Biosciences (Minneapolis, MN). Pannexin-1 polyclonal rabbit antibodies were purchased from Alomone (Jerusalem, Israel), and Connexin-43 (Cx43) polyclonal rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluo-3AM fluorescent dye, Rhodamine-phalloidin and secondary antibodies (Alexa Fluor-conjugated secondary antibody) were purchased from Invitrogen (Carlsbad, CA). CellMask Deep Red Plasma membrane stain was purchased from ThermoFisher (Waltham, MA) and SiR-Actin Spirochrome probe was purchased from Cytoskeleton Inc. (Denver, CO). VectaSHIELD with DAPI was purchased from Vector Labs (Burlingame, CA).
4
P2X7 Receptor Activation Dynamics
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RAW 264.7 cells were seeded in 96-well plates at a density of 4 × 104 cells/well and incubated in DMEM for 24 h, 37 °C, 5% CO2. The cells were then washed twice with HBSS, and the medium was changed to the same buffer containing YO-PRO-1 dye (Sigma, Burlington, MA, USA, 2.5 μM final concentration). Further, the studied compound was added to the cells at concentrations of 5, 1, and 0.1 μM. P2X7 receptor blocker A438079 at a concentration of 10 μM was used as a comparison control. Two mM ATP (Sigma, Burlington, MA, USA) or 200 μM 2,3-O-(4-benzoylbenzoyl)ATP (BzATP, Sigma-Aldrich, St. Louis, MO, USA) were used as an agonist of P2X7R. In some experiments, cells were treated with 350 μM oxidized ATP (OxATP, Sigma-Aldrich, St. Louis, MO, USA) for 2 h. The YO-PRO-1 fluorescence was measured continuously every 100 sec for 800 sec after adding ATP or BzATP with a PHERAstar FS plate reader (BMG Labtech, Ortenberg, Germany) at λex = 480 nm and λem = 520 nm, ATP 2 mM or BzATP 200 μM were added after baseline recording and HBSS (20 μL) was added as a negative control.
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