3T3 cells or A549 cells were cultured on a 96-well plate and transient overexpression of each protein - S1 (Wu, α and δ) - was achieved for 24 h using ProFection Mammalian Transfection System (Promega) or adenovirus infection. Subsequently, cells were induced to take up Fluo 4-AM using Calcium Kit II-Fluo 4 (Dojindo) and then the fluorescence intensity in individual cells was measured using an ArrayScan XTI instrument (Thermo Fisher).
Calcium kit 2 fluo 4
Manufactured by Dojindo Laboratories
Sourced in Japan
The Calcium Kit II-Fluo 4 is a laboratory product developed by Dojindo Laboratories for the detection and measurement of intracellular calcium levels. The kit utilizes the fluorescent indicator Fluo-4, which specifically binds to calcium ions and emits a fluorescent signal upon excitation. This kit provides the necessary components to perform calcium-related experiments and assays.
Automatically generated - may contain errors
Lab products found in correlation
96 well plate, by Iwaki (2 mentions)
Power scan ht, by Agilent Technologies (2 mentions)
Profection mammalian transfection system, by Promega (1 mentions)
Black 96 well plate, by Thermo Fisher Scientific (1 mentions)
Bz x710 fluorescent microscope, by Keyence (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Isogen kit, by Nippon Gene (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Dulbecco s modified eagle medium dmem f12, by Thermo Fisher Scientific (1 mentions)
2 d quant, by GE Healthcare (1 mentions)
Non essential amino acids (neaa), by Cosmo Bio (1 mentions)
Bupropion, by Fujifilm (1 mentions)
Matrigel, by BD (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Lsm 700 laser scanning microscope, by Zeiss (1 mentions)
9 protocols using calcium kit 2 fluo 4
1
Fluorescent Calcium Imaging of Overexpressed Proteins
2
Measuring Intracellular Calcium Dynamics
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The intracellular calcium concentrations were measured by detecting the fluorescence of cells treated with a calcium-sensitive indicator, Fluo-4 AM [17 (link)]. L6 cells harvested 10 days after differentiation were re-plated in the 96-well plates (Iwaki, Tokyo, Japan) at 1.5×104 cells/well for 24 h. Subsequently, the Ca2+ levels were determined with the use of Calcium Kit II-Fluo 4 (Dojindo, Kumamoto, Japan) using Powerscan HT (BioTek, VT, US). Briefly, cells were washed twice with non-serum medium containing 2.5 mM probenecid in 24 h after re-plating. Then cells were incubated with 4 μg/ mL Fluo-4 AM and 0.025% (w/v) Pluronic F-127 for 30 min in dark at 37°C. After washings twice with non-serum medium, cells measurement was performed on a Powerscan HT instrument with an excitation band of 485/20 nm and fluorence was measured at 528/20 nm. Baseline signals (F0) were recorded 5 min before the addition of each stimulus. Subsequently, continuous fluorescence measurements were performed for 20 min. Results are shown as F/F0 ratios after background subtraction, where F was the fluorescence signal intensity and F0 was the baseline intensity, as calculated by the average of 5 frames before stimulus application [17 (link)].
3
Calcium Signaling in HaCaT Keratinocytes
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HaCaT keratinocytes were seeded at a concentration of 2.0 × 105 cells/ml in 100 μl/well in a black 96 well plate (Thermo Fisher Scientific) overnight until confluence. The plate was then washed with PBS (−), and cells were stimulated by incubation with EP concentrations of 0, 50, 100, 500, and 1,000 μM for one hour. Following EP stimulation, the plate was washed again and Calcium Kit II-Fluo 4 (Dojindo) was prepared according to the manufacturer’s instructions, and subsequently added to the cells. Fluorescence intensity was then measured at a wavelength of 485/535 nm in a microplate reader. For groups with DPI pre-treatment, the cells were incubated with 2.5 μM DPI for two minutes and washed prior to 1 mM EP stimulation. For WEB2086 groups, the cells were incubated with 10 μM of the platelet activating factor receptor (PAF-R) antagonist for 10 min and then stimulated with 1 mM EP without washing.
4
Measuring C2C12 Myotube Ca2+ Levels
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Intracellular Ca2+ levels of C2C12 myotubes were measured using a Calcium Kit II-Fluo 4 (Dojindo, Japan) according to the manufacturer’s instructions. Briefly, differentiated C2C12 myotubes in black clear-bottom 96 well plates (Corning, NY, USA) were pre-incubated with 100 μl per well of loading buffer (5% Pluronic F-127, 250-mmol/l Probenecid and 1-μg/μl Fluo 4 AM in Hanks’–HEPES Buffer) for 30 min and subsequently treated with Cy3G for 15–90 min. Fluorescence intensity (excitation/emission 485/528 nm) was then measured using a Powerscan HT plate reader.
5
Intracellular Calcium Imaging in MGN3-1 Cells
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MGN3-1 cells were seeded at 5 × 104 cells/well and cultured overnight in 96-well plates. Intracellular Ca2+ levels ([Ca2+]i) were measured using Calcium Kit II Fluo4 (Dojindo, Kumamoto, Japan). Calcium imaging movies were recorded and analyzed using a BZ-X710 fluorescent microscope (Keyence Corporation, Osaka, Japan) at room temperature (20°C). The mean value of 20 randomly selected cells was calculated.
6
Measuring Intracellular Calcium Dynamics in L6 Cells
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Intracellular calcium concentrations were measured by detecting the fluorescence of cells treated with a calcium-sensitive indicator, Fluo-4 AM [54 (link)]. L6 cells harvested 10 d after differentiation were replated in a 96-well plate (Iwaki, Tokyo, Japan) at 1.5 × 104 cells/well for 24 h. Subsequently, the Ca2+ levels were determined using a Calcium Kit II-Fluo 4 (Dojindo, Kumamoto, Japan) using Powerscan HT (BioTek, VT, USA). Briefly, cells were washed twice with non-serum medium containing 2.5 mM probenecid 24 h after replating. The cells were incubated with 4 μg/mL Fluo-4 AM and 0.025% (w/v) pluronic F-127 for 30 min in the dark at 37 °C. After washing twice with non-serum medium, cells were measured using a Powerscan HT instrument with an excitation band of 485/20 nm, and fluorescence intensity was measured at 528/20 nm. Baseline signals (F0) were recorded 5 min before the addition of each stimulus. Continuous fluorescence measurements were performed for 20 min. The results are shown as F/F0 ratios after background subtraction, where F is the fluorescence signal intensity and F0 is the baseline intensity, as calculated from the average of five frames before stimulus application [54 (link)].
7
Cell Culture and Viability Assay Protocol
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Vs, Hs and Vn were purchased from Sigma Aldrich, USA. Dulbecco’s Modified Eagle Medium (DMEM)/F-12 and Opti-MEM were obtained from Gibco, USA. Fetal bovine serum was from Gibco, South America. Penicillin - Streptomycin were purchased from Biowhittaker, USA. Non-essential amino acids were from Cosmo Bio Co, LTD, Japan. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and dexamethasone were from Dojindo, Japan. Bupropion was from Wako, Japan. ATP bioluminescence kit was from TOYO Ink, Japan. ISOGEN kit was purchased from Nippon Gene, Japan. RIPA lysis buffer was from (Santa Cruz Biotechnology, USA). 2-D Quant was purchased from GE Healthcare Life Sciences, USA. Calcium Kit II-Fluo 4 was from Dojindo, Japan.
8
Calcium-mediated Response to MeHg
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This mobilization induced by MeHg was monitored by means of Calcium kit II -Fluo 4
(Dojindo) as previously described [26 (link), 27 (link)]. Cells in a 96-well plate were incubated with 2.5
µM fluo-4 AM for 1 hr at 37°C. Fluo-4 fluorescence at 518 nm emission
after excitation at 495 nm was measured using the Infinite M200 FA plate reader at
37°C.
(Dojindo) as previously described [26 (link), 27 (link)]. Cells in a 96-well plate were incubated with 2.5
µM fluo-4 AM for 1 hr at 37°C. Fluo-4 fluorescence at 518 nm emission
after excitation at 495 nm was measured using the Infinite M200 FA plate reader at
37°C.
9
Assessing Calcium-Mediated GLP-1 Release in NCIeH716 Cells
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Human enteroendocrine NCIeH716 cells were maintained in suspension culture. Two days before experiments, cells were seeded into 12-well culture plates pre-coated with Matrigel (BD Biosciences, Bedford, MA, USA) as described [21] . On the day of the experiments, supernatants were replaced by PBS containing 1 mM CaCl 2 and DPP-IV inhibitor. The solutions were adjusted to pH 7.2. Cells were incubated for 15 min at 37 C in glucose solutions with or without vitamin D 3 . GLP-1 was measured by GLP-1 ELISA Kit (Sibayagi) and normalized to protein content. Measurement of the intracellular calcium concentration change was performed according to the protocol of Calcium Kit II-Fluo4 (Dojindo, Kumamoto, Japan). Briefly, cells were plated onto Matrigel-coated glass bottom 35 mm dishes and were loaded with fluo4 at 37 C. Following the 1 h loading period, cells were washed twice with HKRB buffer (20 mM HEPES, 103 mM NaCl, 4.77 mM KCl, 0.5 mM CaCl 2 , 1.2 mM MgCl 2 , 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , 15 mM glucose, pH 7.3). Confocal imaging was performed using the LSM 700 Laser Scanning Microscope (Zeiss) using a 60 Â oil objective. Images were acquired and processed with ZEN 2012 software (Zeiss).
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