A minimum of 1 × 105 AM were plated in a 35 mm dish and incubated for 2 h. The cells were prepared by incubation for 30 min in 1 ml loading buffer containing Ringer solution (140 mM NaCl, 5 mM KCl, 5 mM CaCl2, 2.5 mM MgCl2, 1 mM HEPES) and 7.5 μM Fura-2 AM (Enzo, Farmingdale, USA, cat. ENZ-52006) like described before (Gelis et al. 2016 (link)). After removal of the extracellular Fura-2 AM by washing with Ringer solution, Ca2+-ratiometric imaging was performed using a light source (EL6000, Leica), a Leica inverted confocal microscope (DMI6000 CS, Leica) with a 20× objective (UPLSAPO, Olympus, Tokyo, Japan). The images were recorded at 1 Hz by the DFC360 FX (Leica, Wetzlar, Germany), and the integrated fluorescence (f 340 nm/f 380 nm) of each cell was measured using the Leica Application Suite Advanced Fluorescence (LAS AF). The odorants were pre-diluted in DMSO and then adjusted to the final concentration in Ringer solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCL2, and 10 mM Hepes). The inhibitors MDL-12,330A (10 µM; Sigma-Aldrich, St. Louis, USA, cat: M182) and Oxyphenylon (300 µM; Henkel) were also pre-diluted in DMSO. EGTA (10 mM; Sigma-Aldrich, St. Louis, USA, cat: 03777) was prepared in water.
Fura 2 am
Manufactured by Enzo Life Sciences
Sourced in United States
Fura-2/AM is a cell-permeant fluorescent calcium indicator. It is used to measure intracellular calcium concentrations in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Sodium phosphate monobasic, by Avantor (2 mentions)
D glucose, by Avantor (2 mentions)
Nis elements software, by Nikon (2 mentions)
El6000, by Leica (1 mentions)
Dfc360 fx, by Leica (1 mentions)
Dmi6000 cs, by Leica (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (1 mentions)
Application suite advanced fluorescence las af, by Leica (1 mentions)
Oxyphenylon, by Henkel Adhesive Technologies (1 mentions)
Uplsapo, by Olympus (1 mentions)
Mdl 12 330a, by Merck Group (1 mentions)
Fura 2 am, by Leica (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
Verapamil, by Merck Group (1 mentions)
Ag1 x8 resin, by Bio-Rad (1 mentions)
O phthaldialdehyde opt, by Merck Group (1 mentions)
3h inositol, by PerkinElmer (1 mentions)
U46619, by Merck Group (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Alexa flour 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
6 protocols using fura 2 am
1
Fura-2 AM Calcium Imaging Protocol
2
Fatty Acid and Signaling Compound Analysis
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C18 fatty acids (stearic acid, oleic acid, linoleic acid and α-linolenic acid), verapamil, 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), bisindolylmaleimide (BIM), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and o-phthaldialdehyde (OPT) were obtained from Sigma Chemical (St. Louis, MO, USA). Fura-2/AM was purchased from Enzo Life Science (Farmingdale, NY, USA). [3H] Inositol was obtained from PerkinElmer (Waltham, MA, USA). AG 1-X8 resin was purchased from BIO-RAD (Hercules, CA, USA). The materials for cell culture were purchased from Life Technologies (Grand Island, NY, USA). Unless otherwise stated, all reagents were of the highest purity and were purchased from Sigma Chemical.
3
Synthesis and Characterization of HPW-RX40
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HPW-RX40 (2-Methoxy-4-[(E)-2-nitrovinyl]phenyl 2,3-dichlorobenzoate) was synthesized according the methods described previously [17] (link). Bovine α-thrombin was purchased from Biovision, (Mountain View, CA, USA). Collagen (Type I, equine tendon) was from Helena Laboratories (Beaumont, TX, USA). Fibrinogen, U46619 (9,11-dideoxy-9,11-methanoepoxy PGF2α), 12-O-tetradecanoylphorbol-13-acetate (TPA), A23187, eosin isothiocyanate, oxidized glutathione (GSSG), and phenyl arsenoxide (PAO) were from Sigma-Aldrich (St. Louis, MO, USA). Alexa Flour 488-conjugated phalloidin, was obtained from Molecular Probes (Eugene, OR, USA). Thapsigargin and rutin were purchased from Cayman Chemical (Ann Arbor, MI, USA). Human recombinant PDI, ERp72, and Fura-2/AM were purchased from Enzo Life Sciences (Farmingdale, NY, USA); Human recombinant ERp57 and anti-P47 Ab were from Abcam (Cambridge, UK), and ERp5 was from ProSpec Protein Specialists (Rehovot, Israel). Anti-PDI Ab was from Pierce/Thermo (Rockford, IL, USA); anti-GRP78 Ab was from Genetex (Irvine, CA, USA); phospho-PKC substrate antibody and anti-CHOP Ab were from Cell Signaling Technology (Beverly, MA, USA); anti-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were purchased from Sigma Chemical Co.
4
Fura-2 AM Calcium Imaging Protocol
5
Calcium Imaging of DRG Neurons
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For Ca2+ imaging experiments, cultured DRG neurons for 24‐48 hours were incubated for 30 minutes in DMEM containing 1 μmol/L of the fluorescent indicator Fura‐2 AM (Enzo Life Sciences, Inc, NY). Fura‐2 fluorescence was measured under conditions where a coverslip was set in a recording chamber that was then perfused with a standard bath solution containing 140 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L MgCl2, 2 mmol/L CaCl2, 10 mmol/L Hepes, and 10 mmol/L d ‐glucose at pH 7.4 adjusted with NaOH. Fura‐2 fluorescence was excited at 340 and 380 nm, and emission was monitored at 510 nm with a digital CCD camera (Andor Co., Ltd, London, UK). Data were obtained every 5 seconds using a software (NIS‐Elements AR 4.13.04 64‐bit, Nikon Corporation, Japan) and analyzed using Microsoft Excel (Microsoft, WA, USA). The 340/380 ratio value (described as F) was calculated using regions of interest that included the whole cell body of single DRG neurons. The changes in the 340/380 ratio values (F/F0) were calculated using F0, averaged F value for the first 25 seconds of respective DRG neurons in experiments. In this study, changes in each response to an application with F/F0>0.1 were regarded as positive. Cells not responding to 100 mmol/L KCl, which was applied in the last of each experiment, were regarded as non‐neuronal cells and excluded from analysis.
6
Fura-2 AM Calcium Imaging Protocol
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Before imaging, cells were incubated in 350μL of complete media containing 5μM Fura-2 AM (Enzo) for 30 min at 37°C with 5%CO2. During imaging, the cells were perfused with a buffer containing the following: 135mM sodium chloride, 3.2mM potassium chloride, 2.5mM magnesium chloride, 2.8mM calcium chloride, 667μM monobasic sodium phosphate, 14.2mM sodium bicarbonate, and 10.9mM D-glucose (all from VWR) with a pH between 7.00 and 7.40. The buffer and the holding plate were kept at 37°C while imaging. Imaging data was collected on a TE200 inverted microscope using NIS Elements software (Nikon). Cells were exposed to 340 and 380nm wavelengths for 100 ms and the A340/A380 ratio was calculated. Traces were analyzed using Excel and responses greater than 10% of the baseline were counted. Each data point in the scatterplots represents one coverslip.
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