Cells were seeded on gelatin-coated glass coverslips at a density of 5000 cells/cm2 at 24 h before the experiments. Cells were starved in DMEM 5% FBS for at least 2 h before the experiments. Cells were next loaded (45 s at 37 °C) with 2 μM Fura-2 AM, for ratiometric cytosolic calcium concentration ([Ca2+]i) measurements. During experiments, cells were maintained in standard extracellular solution of the following composition: 154 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 5.5 mM glucose. Solution pH was adjusted to 7.35 using NaOH. 4 h before experiments cells were starved in DMEM 2% FBS. Fluorescence measurements were made using a Polychrome V spectrofluorimeter (TILL Photonics, Munich, Germany) attached to an Olympus ×51 microscope (Olympus, Tokyo, Japan) and Metafluor Imaging System (Molecular Devices, Sunnyvale, CA, USA). [Ca2+]i was measured using ratiometric probe Fura-2-AM and quantified according to Fiorio Pla et al. [50 (link)].
Metafluor imaging system
Manufactured by Molecular Devices
Sourced in Japan
The Metafluor Imaging System is a high-performance microscopy platform designed for fluorescence imaging. It provides versatile capabilities for a wide range of biological and chemical applications. The system utilizes advanced optics and imaging technologies to capture and analyze fluorescent signals with precision and accuracy.
Automatically generated - may contain errors
3 protocols using metafluor imaging system
1
Fura-2 Calcium Imaging Protocol
2
Calcium Imaging in A549 Cells
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A549 cells were plated on coverslips and incubated with 5 µM Fura-2 AM (cat. no. F1221; Invitrogen; Thermo Fisher Scientific, Inc.) in physiological salt solution (PSS) at 37°C for 60 min, then washed with PSS with or without antagonist for 20 min. Cells on coverslips were then mounted in a standard perfusion chamber on a Nikon microscope table. The fluorescence ratio of Fura-2 (F340/380) was tracked over time at an excitation wavelength of 340 or 380 nm and captured using an intensified CCD camera (Hamamatsu Photonics K.K.) and a MetaFluor Imaging system (Version 7.10.4.407; Molecular Devices, LLC). The ratio of F340/380 represented the intracellular Ca2+ levels and was quantified using Δ(F340/380), that is, the difference between the baseline and maximum values after stimulation. The PSS for Ca2+ measurement consisted of the following: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM HEPES and 10 mM glucose at pH 7.3. The osmolality of the solution was ~300 mOsmol/kg H2O.
3
Measuring Intracellular Calcium in Immune Cells
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Cells (PECs) were loaded with 2 μM of Fura-2-acetoxymethyl ester (Fura-2/AM, Life Technologies), 0.5% BSA in the recording saline solution. After 1 h, cells were stimulated for 2 min with LPS (1 μg/mL). When indicated, cells were also pre-incubated with PGB (11.3 μM) for 15 min. Intracellular Ca2+ concentration ([Ca2+]) was assessed by recording the changes in cytoplasmic [Ca2+] with the ratiometric fluorescent probe Fura-2 in PECs. The MetaFluor Imaging System (Molecular Devices) was used for fluorescence acquisition and analysis of individual cells. Pairs of images were acquired every 2 s. A single PEC was considered as a responder if the F340/F380 ratio for a single PECincreased by 0.05.
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