Fluo-5F and Fluo-4 pentapotassium salt and Alexa Fluor 594 hydrazide Na salt were from Molecular Probes. UBO-QIC (Gαq-inhibiting compound) was from the Institute of Pharmaceutical Biology, University of Bonn, Germany. 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), gabazine (SR95531), (-)-Quinpirole hydrochloride, (S)-(-)-sulpiride, phorbol 12-myristate 13-acetate (PMA), U0126, cyclopiazonic acid (CPA), thapsigargin, heparin sodium salt, ryanodine, U73122, 8-Br-cyclic ADP ribose, tetrodotoxin-citrate, pertussis toxin, and nifedipine were from Tocris. All others were from Sigma. All drugs were introduced to the artificial cerebrospinal fluid unless otherwise noted.
Fluo 5f
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
Fluo-5F is a fluorescent calcium indicator used for the detection and measurement of intracellular calcium levels in cells. It is a cell-permeant, green-fluorescent dye that exhibits an increase in fluorescence upon binding to calcium ions.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 594, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (4 mentions)
Cgp55845, by Bio-Techne (3 mentions)
Picrotoxin, by Bio-Techne (3 mentions)
Mni caged l glutamate, by Bio-Techne (2 mentions)
Dcg 4, by Bio-Techne (2 mentions)
D apv, by Bio-Techne (2 mentions)
Quinpirole, by Bio-Techne (2 mentions)
Thapsigargin, by Bio-Techne (1 mentions)
Nifedipine, by Bio-Techne (1 mentions)
Quinpirole hydrochloride, by Bio-Techne (1 mentions)
Heparin sodium salt, by Bio-Techne (1 mentions)
Ryanodine, by Bio-Techne (1 mentions)
Pertussis toxin, by Bio-Techne (1 mentions)
U73122, by Bio-Techne (1 mentions)
Tetrodotoxin citrate, by Bio-Techne (1 mentions)
S sulpiride, by Bio-Techne (1 mentions)
Fluo 4 pentapotassium salt, by Thermo Fisher Scientific (1 mentions)
Phorbol 12 myristate 13 acetate, by Bio-Techne (1 mentions)
U0126, by Bio-Techne (1 mentions)
22 protocols using fluo 5f
1
Neurochemical Signaling Pathway Reagents
2
Purification and Reconstitution of SERCA
3
Imaging and Uncaging for Neuroscience
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Fluo-5F and Alexa Fluor 594 were from Molecular Probes. MNI-caged-L-glutamate, Ifenprodil, Ro 25-6981 and TTX were purchased from Tocris (Ellisville, MO). NVP-AAM077 was from Sigma. Imaging dyes and MNI-caged-L-glutamate/blockers were introduced to the pipette and the artificial cerebrospinal fluid, respectively.
4
Intracellular Calcium Dynamics Imaging
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Twenty-four hours prior to Ca2+ imaging, the cells were transferred to glass-bottom dishes while diluted 1:6 (~1 × 105 cells). Fluo-5F (Kd = 2.3 μM) (Invitrogen) was used for measuring intracellular Ca2+ concentration [Ca2+]i. The cells were loaded at room temperature for 30 min with Fluo-5F (1 μM) dissolved in the perfusion buffer (KRB) supplemented with 5 mM glucose. Stimulation was carried out by 16.7 mM glucose KRB buffer in the absence or presence of DMSO (1:1000) or Bay K8644 (300 nM) or isradipine (2 μM), and/or 70 mM KCl KRB buffer at room temperature. Time lapse region of interest (ROI) images, the mean and peak intensity of ROIs were acquired by confocal microscopy using a ×40 water immersion objective. A ratio was calculated by taking the fluorescence intensity in the time lapse divided by the average fluorescence intensity under pre-stimulatory conditions. The frequency of peak intensity was counted as ratio >1.5, and the time integral of the fluorescence signal (AUC) was calculated by GraphPad software.
5
Detailed Compound Acquisition Procedure
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All chemicals and drugs used were purchased from MilliporeSigma (St. Louis, MO, USA) except D-APV, NBQX, CGP-55845, DCG-IV, MNI-caged-L-glutamate and CPA which were obtained from Tocris Bioscience (Minneapolis, MN, USA), LY 303070 which was obtained from ABX advanced biochemical compounds (Radeberg, Germany), and Fluo5f and Alexa which were purchased from Invitrogen (Carlsbad, CA, USA).
6
Dual TIRF Imaging of Cav1.2 Sparklets
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We recorded Cav1.2-mediated Ca2+ sparklets using the dual TIRF imaging/patch clamp system described above. HEK293T cells transfected with untagged mouse Cav1.2, pDsRed-monomer-C1 or DsRed-Kv2.1P404W, Cavβ3, Cavα2δ1, and rat PKCα (Navedo et al., 2006 (link)), which increases spontaneous sparklet activity, were loaded via the patch pipette with a solution containing (in mM): 0.2 Fluo-5F (Invitrogen Cat# F14221), 87 Cs-aspartate, 20 CsCl, 1 MgCl2, 5 Mg∙ATP, 10 HEPES, 10 EGTA, adjusted to pH 7.2 with CsOH. After obtaining a GΩ seal in KRB, the external solution was exchanged with a solution containing (in mM): 110 NaCl, 5 CsCl, 1 MgCl2, 10 glucose, 10 HEPES, 20 CaCl2, pH 7.4 with NaOH. Cells were maintained at a holding potential of −70 mV, and TIRF images were acquired using TILLvisION software. Sparklets were manually detected and analyzed using Fiji software. Sparklet activity was quantified by calculating the nPs of each site (Navedo et al., 2006 (link)).
7
Two-Photon Calcium Imaging of Neuronal Activity
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Calcium imaging experiments were performed with a two-photon random-access microscope using acousto-optic deflector (AOD)-based scanning, as described previously (Otsu et al., 2014 (link)). Whole-cell patch pipettes were filled with an intracellular solution containing 135 mM CsMeSO3, 4.6 mM MgCl2, 10 mM HEPES, 10 mM K2-creatine phosphate, 4 mM Na2-ATP, 0.4 mM Na2-GTP, and 2 mM QX-314, pH 7.35 with CsOH (∼300 mOsm) and supplemented with a morphological (10 μM Alexa 594, Invitrogen) and a calcium-sensitive dye (500 μM Fluo-5F, Invitrogen). Cells were voltage-clamped at −60 mV in the whole-cell configuration at 32°C–34°C in the continuous presence of 50 μM D-(2R)-amino-5-phosphonovaleric acid (D-APV), 5 μM SR95531, 1 μM CGP55845, 0.5 μM CGS15943, and 0.25 μM LY341495. Calcium signals were acquired for 100–200 ms before repeated extracellular stimulation (5 pulses, 100 Hz) of the incoming PS input and for 1.8–2 s following stimulation onset. Dwell times of 20–30 μs per point were used. Stimulation-induced calcium transients were acquired every 15 s for 10 or 15 min before applying NASPM (20–50 μM). For data analysis, relative fluorescence was expressed as ΔG/R; i.e., variations in Fluo-5F signal change (ΔG) divided by calcium-independent Alexa 594 red fluorescence (R) (Otsu et al., 2014 (link)).
8
Two-Photon Calcium Imaging of Granule Cells
9
Measuring Intracellular Calcium Dynamics
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A low-affinity Ca2+ indicator, Fluo-5F (Kd=2.3 μM) (Invitrogen), was used for measuring intracellular Ca2+. Cells were loaded with 1 mM Fluo-5 F for 30 min at 37 °C. Cells were first perfused in a Krebs-Ringer bicarbonate (KRB) buffer containing 116 mM NaCl, 4.7 mM KCl, 2.6 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 20 mM NaHCO3, 16 mM HEPES and 2 mg ml−1 BSA, supplemented with 2.8 mM glucose. The cells were stimulated with a 20 mM glucose KRB buffer at room temperature.
Images were acquired by confocal microscopy (Carl Zeiss, Germany) using a × 63 oil immersion objective (NA=1.25). An argon laser (488 nm) was used to excite the cells and the emitted light was collected using a 500–530 nm bandpass filter. The ratio of fluorescence intensity at each time point (Fi) versus the average fluorescence intensity (F0) under pre-stimulatory conditions was determined49 (link). The integrated fluorescence signal (area under the curve) was calculated using the function , where Δt denotes the time interval and Ri the ratio (Fi/F0) at time point i.
Images were acquired by confocal microscopy (Carl Zeiss, Germany) using a × 63 oil immersion objective (NA=1.25). An argon laser (488 nm) was used to excite the cells and the emitted light was collected using a 500–530 nm bandpass filter. The ratio of fluorescence intensity at each time point (Fi) versus the average fluorescence intensity (F0) under pre-stimulatory conditions was determined49 (link). The integrated fluorescence signal (area under the curve) was calculated using the function , where Δt denotes the time interval and Ri the ratio (Fi/F0) at time point i.
10
Pharmacological Modulation of Neural Signaling
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Picrotoxin, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), trans-2-Carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydro-quinoline (L-689560), Nimodipine, Mibefradil, apamin, TTX, DHPG, MPEP and 6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide (YM298198) hydrochloride were purchased from Tocris. Fluo-5F and Alexa Fluor 594 were purchased from Invitrogen. All other chemicals were purchased from Fisher Scientific.
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