CHO cells were transfected with a plasmid expressing mouse Mrgprd (OriGene Technologies, Rockville, USA) with GeneJuice Transfection Reagent (1 μg of plasmid for 3 μL of GeneJuice). The cells were incubated in Ham’s F12 Nutrient Mixture with 5% of FBS. G418 (Sigma) was used as the selection antibiotic. Cells (50×103 cells/well) in a 96-well plate were incubated with fluo-8 loading solution (Fluo-8-AM; Invitrogen) according to the manufacturer’s instructions. The fluorescence was then measured at 530 nm on a microplate reader (NOVOstar; BMG Labtech) for 1 min. Five seconds after the beginning of the calcium measures, 5-oxoETE (1, 10, 25, 50, 100 and 200 μM) or β-alanine (Sigma-Aldrich; 1 mM) was added. Data were collected and analyzed with the NOVOstar software.
Fluo 8 am
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluo-8 AM is a fluorescent calcium indicator dye that can be used to measure intracellular calcium levels in living cells. It functions by undergoing a change in fluorescence intensity upon binding to calcium ions.
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Lab products found in correlation
Pluronic f 127, by Thermo Fisher Scientific (6 mentions)
Eclipse e600fn, by Nikon (2 mentions)
Coolsnap es, by Teledyne (2 mentions)
Lambda ls, by Sutter Instruments (2 mentions)
Lsm 780, by Zeiss (2 mentions)
β alanine, by Merck Group (1 mentions)
Novostar, by BMG Labtech (1 mentions)
Fluo 8 am, by Abcam (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Tcs sp5 confocal laser system, by Leica camera (1 mentions)
Fluoview viewer software, by Olympus (1 mentions)
Fluorodishes, by World Precision Instruments (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Glucose, by Merck Group (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Leica application suite, by Leica camera (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Pluronic acid f 127, by Merck Group (1 mentions)
13 protocols using fluo 8 am
1
Measuring Calcium Flux in CHO Cells
2
Fluo-8 Fluorescent Ca2+ Imaging
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Fluo-8/AM (Abcam, Shanghai, China) is a widely used fluorescent Ca2+ indicator. Before binding to Ca2+, Fluo-8 produces little florescence. However, after Fluo-8 combines with Ca2+, the fluorescence intensity increases at least 200 times. Fluo-8 is excited by light at a wavelength of 488 nm using an argon ion laser, and the emitted light is at a wavelength of 514 nm. We incubated HUVECs for 20 min at 37 °C with 2 μM Fluo-8/AM and 0.02% pluronic F-127 (Invitrogen, Eugene, OR, USA). After the cells were washed with phosphate-buffered saline (PBS) to remove extracellular Fluo-8 dye, the cellular Ca2+ store was depleted by the application of thapsigargin (TG, 2 μM) in a Ca2+-free solution (OPSS, 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 5 mM HEPES, pH 7.3 to 7.4 adjusted with NaOH). The Ca2+ influx was initiated by applying 2 mM Ca2+ to the medium. The baseline fluorescence intensity before extracellular Ca2+ application was considered F0. The peak fluorescence intensity after extracellular Ca2+ application was considered F1. The change in Ca2+ concentration in the cells is presented as a fluorescence intensity ratio (F1/F0) before and after the addition of extracellular Ca2+.
3
Measuring Intracellular Calcium Dynamics
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The [Ca2+]i was measured as previously described (Du et al., 2012 (link)). Briefly, ASM cells were incubated with 6 μmol/L Fluo-8 AM (a medium affinity green fluorescent Ca2+ binding dye) and 0.02% pluronic F-127 (Invitrogen) for 30 min. The cells were treated with 1 nM GSK1016790A with or without 10 μM HC067047. Fluorescence was recorded using a fluorescence microscope (Nikon, Tokyo, Japan) having a xenon lamp with excitation and emission wavelengths of 488 nm. The [Ca2+]i changes are presented as the ratio of the experimental fluorescence relative to the initial fluorescence intensity (F1/F0).
4
Measurement of Cytosolic Calcium Dynamics
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Cytosolic Ca2+ ([Ca2+]i) was measured as previously described (Shen et al. 2011 ). In brief, cells were incubated with 10 μmol/L Fluo‐8/AM and 0.02% pluronic F‐127 (Invitrogen, Carlsbad, CA) for 40 min in the dark at 37°C. Ca2+ stores were depleted by treating cells with 4 μmol/L TG for 10 min in 0Ca2+‐PSS. Ca2+ influx was initiated by applying 1 mmol/L extracellular Ca2+. Cells were pretreated with scrambled siRNA or Orai1 siRNA for 24 h before experiments. Fluorescence signal was recorded by Leica TCS SP5 confocal laser system. Changes in [Ca2+]i were displayed as the ratio of fluorescence relative to the intensity before applying extracellular Ca2+ (F1/F0).
5
Measuring Calcium Signaling in Cells
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Calcium signalling was performed as previously described [19 (link), 22 (link)]. Briefly, cells were grown until ∼80 % confluency on glass-bottomed FluoroDishes (World Precision Instruments, Sarasota, FL, USA). Cells were serum starved for 3 h and pre-treated for 1 h with S1PR1 antagonist (NIBR-0213) [23 (link)] or left in serum-free media. The cells were washed with 1 ml 37 °C HBSS (Invitrogen) supplemented with 20 mM HEPES buffer (Invitrogen) and 5.5 mM glucose (Sigma Aldrich). Cells were then loaded with 2 μM Fluo-8 AM (Invitrogen) in supplemented 37 °C HBSS for 40 min at 37 °C and 5 % CO2. Fluo-8 AM dye was removed and cells were washed with 37 °C supplemented HBSS. Next, cells were left to rest in 1 ml supplemented HBSS at room temperature in the dark for 20 min. Calcium responses were recorded using an Olympus FV1000 Confocal Microscope with ×20 lens. For analysis, images were obtained at a rate of 1 frame/2 s for a total of 250 s. Images were then analysed using the Olympus Fluoview viewer software. Fluorescence was normalised to mean baseline fluorescence (0–30 s) (ΔF/F0). GraphPad Prism 4 software was used to generate calcium response traces presented as ΔF/F0 over time.
6
Measuring Intracellular Calcium Dynamics in HUVECs
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[Ca2+]i measurements were performed as previously described (20 (link)). Briefly, HUVECs were seeded on round glass cover slips placed in 12-well plates and treated with PTH (100 pM) for 24 h before measurement. The cells were incubated for 20 min at 37°C with 2 μM Fluo-8/AM and 0.02% pluronic F-127 (Invitrogen) in the culture media. Intracellular Ca2+ stores were depleted using 2 μM TG in a Ca2+-free saline solution (OPSS, 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 5 mM HEPES, pH 7.3 to 7.4 adjusted with NaOH). Application of 2 mM Ca2+ to the medium evoked Ca2+ influx. The [Ca2+]i is shown as fluorescence signals and was measured using a fluorescence microscope (Nikon T200, Tokyo, Japan). The baseline before the application of extracellular Ca2+ was considered F0, and [Ca2+]i changes are expressed as the ratio of fluorescence relative to the intensity at baseline (i.e., F1/F0).
7
Calcium Imaging of ARPE-19 Cells
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For Ca2+ imaging studies, ARPE-19 cells at confluence were plated onto round coverslips covered with poly-l -lysine (10 μg/ml, Sigma Chemical) and incubated in the dark with 10 μM fluo 8-AM (Invitrogen) dissolved in the extracellular buffer containing: 150 mM NaCl, 20 mM HEPES, 0.1 mg/ml bovine serum albumin, 1.0 mM EGTA, adjusted to pH 7.4 with NaOH. Loaded cells were transferred into a perfusion chamber located on a microscope adapted to an epifluorescence system (Eclipse E600FN; Nikon Melville, NY). Cells were constantly perfused with oxygenated extracellular buffer at a rate of 15–17 ml/min and at 29–30 °C. Excitation of the fluorophore (at 488 nm) was performed with a light source controlled by a Lambda LS illuminator (Sutter Instruments, Novato, CA). Images were acquired with a cooled digital camera (Cool-SNAP-ES, Roper Scientific, Tucson, AZ) using the RS image software (Photometrics; Roper Scientific). The image field was 800 × 600 μm in size (Ramirez et al., 2012 (link)). Short movies (180 s total time, at 40 ms exposure at 4 Hz) were taken in control conditions and during the wash-in of the pharmacological treatments. To normalize the change in fluorescence of the treated cultures, their Δ fluorescence was corrected by subtracting that of one of the untreated conditions.
8
Calcium Imaging of TC28a2 Cells
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TC28a2 cells were cultured overnight at a density of 30,000 cells per well in a 24-well plate for image analysis or 10,000 cells per well in a 96-well plate for quantification using GloMax®-Multi+ Detection System (Promega, Madison, WI, USA). Cells were then serum starved overnight. Fluo8AM (Invitrogen) was diluted to 5 mM in DMSO. The Fluo 8AM was then diluted to 5 µM in DMEM before addition to cells. Cells were incubated in 5 µM Fluo8 for 30 min before washing with DMEM three times. Cells were incubated for a further 30 min prior to fluorescent imaging with the Leica DFC365 FX microscope with the Leica Application Suite (Leica Wetzlar, Germany) or fluorescence detection on the GloMax®-Multi+ Detection System (Promega).
9
Measuring Calcium Influx in Endothelial Cells
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HCAECs were plated on 30 mm glass cover slips. The changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured after HCAECs were treated with the HG medium. HCAECs were incubated with 6 µM Fluo-8 AM and 0.02% Pluronic F-127 (Invitrogen) for 30 min in an incubator. Subsequently, the Ca2+ stores were depleted by treatment with 2 µM thapsigargin (TG) or 100 µM ATP for 10 min in a Ca2+-free saline solution (in mM: NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 11.1, at pH 7.4). Ca2+ influx was induced by the application of 2 mM extracellular Ca2+. Fluorescence was detected and recorded using a fluorescence microscope (Nikon T200, Tokyo, Japan) having a xenon lamp with excitation and emission wavelengths of 488 and 515 nm, respectively. The [Ca2+]i changes were analyzed as the ratio of the fluorescence intensity after the extracellular Ca2+ addition relative to the intensity before the application of extracellular Ca2+ (F1/F0).
10
Visualizing Calcium Dynamics in Hippocampal Slices
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PFC slices were incubated at room temperature, in the dark, for 2 h in the presence of 10 µM Fluo-8 AM (Invitrogen) and 0.3% pluronic acid in aCSF equilibrated with carbogen [37 (link), 39 (link)–41 (link)]. Then, after a recovery period of 2 h, the slices were transferred and immobilized, with a nylon mesh, into a perfusion chamber on a microscope adapted to an epifluorescence system (Eclipse E600FN; Nikon, Melville, NY). Slices were continuously perfused with aCSF equilibrated with carbogen at 30–32°C. Excitation at 488 nm was performed with a Lambda LS illuminator (Sutter Instrument, Novato, CA), and images were acquired with a cooled digital camera (CoolSNAP-ES, Roper Scientific, Tucson, AZ). The imaging software used was RS Image (Photometrics; Roper Scientific, Tucson, AZ), and the imaged field was 800 × 600 µm. Short movies (175 s, 40-µs exposure, four images per second) were taken. Cells active during the experiment were analyzed. The hippocampal axonal bundle was stimulated electrically as described above in control conditions and in the presence of Aβ.
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