Intracellular Ca2+ levels in VSMCs were assessed using the fluorescent Ca2+ indicator Cal-520 acetoxymethyl ester (Cal-520AM; Abcam, Cambridge, U.K.). Quiescent VSMCs cultured in 12-well plates were incubated with 10 μM Cal-520AM in DMEM containing 0.5% FBS at 37°C for 75 min, followed by 30 min at room temperature. Subsequently, the dye solution was replaced with HEPES buffer (130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM d -glucose and 20 mM HEPES, pH 7.4) for 30 min. In some experiments, inhibitors such as gefitinib (1 µM), NS8593 (40 µM) and 2-APB (30 µM) were added 30 min prior to stimulation with EGF (50 ng/ml). Fluorescent Cal-520AM-Ca2+ signals were recorded by an inverted epifluorescence microscope (Axio Observer Z1 Live-Cell imaging system, ZEISS, Cambridge, U.K.) at excitatory wavelength of 490 nm and emission of 535 nm. Data were acquired and analyzed using Zen Pro (ZEISS).
Axio observer z1 live cell imaging system
Manufactured by Zeiss
Sourced in United Kingdom
The Axio Observer Z1 is a live-cell imaging system manufactured by Zeiss. It is designed for long-term observation and analysis of living cells. The system provides a controlled environment for the sample, maintaining optimal conditions for cell culture. It features high-resolution optics and a range of imaging modalities, enabling detailed observation of cellular processes.
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5 protocols using axio observer z1 live cell imaging system
1
Intracellular Ca2+ Dynamics in VSMCs
2
Real-time TRPM7 dynamics in HEK-293
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HEK-293 expressing yellow fluorescent protein (YFP)-tagged WT mouse TRPM7 (WT mTRPM7-YFP) [41 (link)]. The transient transfected HEK-293 cells were maintained in DMEM containing 10% FBS. After EGF (50 ng/ml) treatment, fluorescent signal of TRPM7 in HEK-293 cells were recorded for 15 min on an inverted epifluorescence microscope (Axio Observer Z1 Live-Cell imaging system, ZEISS) at excitatory wavelength of 490 nm and emission of 535 nm. Images were quired and analyzed by Zen Pro software (ZEISS).
3
VSMC Calcium Signaling Imaging Protocol
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VSMC Ca2+ signaling was assessed using the fluorescent Ca2+ indicator, Cal‐520 acetoxymethyl ester (Cal‐520/AM; 10 μmol/L; Abcam, Cambridge, UK). Cells were grown in 12‐well plates and following removal of culture media were incubated with Cal‐520 AM in 0.5% FBS at 37 °C for 75 minutes followed by 30 minutes at room temperature. Following incubation, the dye solution was replaced with HEPES physiological saline solution (1.3×10−1 mol/L NaCl, 5×10−3 mol/L KCl, 10−3 mol/L CaCl, 10−3 mol/L MgCl, 2×10−2 mol/L HEPES, and 10−2 mol/L D‐glucose, pH 7.4) for 30 minutes prior to imaging. Fluorescence intensity as a measure of [Ca2+]i, was monitored for 30 seconds in basal condition and 180 minutes under endothelin‐1 (0.1 μmol/L) or U46619 (1 μmol/L) stimulation. In some experiments, VSMCs were pretreated for 30 minutes with olaparib (1 μmol/L) or 8‐Br‐cADPR (1 μmol/L). Fluorescence‐based measurements of Ca2+ signals were performed using an inverted epifluorescence microscope (Axio Observer Z1 Live‐Cell imaging system; Zeiss, Cambridge, UK) with excitation/emission wavelengths 490/535 nm, respectively. Images were acquired and analyzed using the Zen Blue Program (Zeiss, Cambridge, UK).
4
Angiotensin II-Induced VSMC Ca2+ Signaling
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VSMC Ca2+ signaling was assessed using the fluorescent Ca2+ indicator, Cal-520 acetoxymethyl ester (Cal-520 AM; Abcam; 5 × 10–6 mol/L). Cells were grown in 6-well plates and following removal of culture media were incubated with 10–5 mol/L Cal-520 AM in 0.5% FBS at 37°C for 75 minutes followed by 30 minutes at room temperature. Following incubation, the dye solution was replaced with HEPES physiological saline solution (1.3 × 10–1 mol/L NaCl, 5 × 10–3 mol/L KCl, 10–3 mol/L CaCl2, 1 × 10–3 mol/L MgCl2, 2 × 10–2 mol/L HEPES, and 1 × 10–2 mol/L D-glucose, pH 7.4) for 30 minutes prior to imaging. VSMCs were stimulated with Ang II (1 × 10–7 mol/L) and the Ca2+ transient tracked for 5 minutes. In some experiments, VSMCs were pretreated for 24 hours with 4-PBA or fasudil. Fluorescence-based measurements of Ca2+ signals were performed using the inverted epifluorescence microscope Axio Observer Z1 Live-Cell imaging system (Zeiss) with excitation/emission wavelengths 490 nm and 525 nm, respectively. Images were acquired and analyzed using Zen Pro (Zeiss).
5
Measurement of Intracellular Calcium Levels
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Intracellular free Ca2+ levels were measured in RAEC using the fluorescent Ca2+ indicator, Cal-520 acetoxymethyl ester (Cal-520/AM; Abcam; 10 μmol/l). Fluorescence measurements were performed using an inverted epifluorescence microscope (Axio Observer Z1 Live-Cell imaging system; Zeiss) with excitatory wavelengths of 490 and emission of 535. Images were acquired and analysed using Zen Blue Program (Zeiss). Cells were grown in six-well plates and following the removal of culture media were incubated with 10 μmol/l of Cal-520 AM in 0.5% FBS at 37°C for 75 min followed by 30 min at room temperature. Following incubation, the dye solution was replaced with HEPES physiological saline solution containing the following components (in mmol/l): NaCl 130, KCl 5, CaCl 1, MgCl 1, HEPES 20 and D-glucose 10, pH 7.4) for 30 min before imaging. Fluorescence intensity as a measure of [Ca2+]i, was monitored for 30 s in basal condition and 2.5 min under Ang II 10−7 mol/l stimulation.
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