For intracellular Ca2+ ([Ca2+] i) determination, PASMCs or PAECs cultured on 35‐mm dishes were incubated in a culture medium containing 3.5 μmol/L FURA‐2‐AM (Invitrogen) for 1 hours at 37°C and subsequently rinsed with HBSS (Sigma). Each dish was placed into a culture chamber at 37°C on the stage of an inverted fluorescence microscope (NikonTE2000E), connected to a cooled CCD camera (512B Cascade, Roper Scientific). Samples were illuminated alternately at 340 and 380 nm using a random‐access monochromator (Photon Technology International), and emission was detected using a 510 nm emission filter. Images were acquired (1 ratio image per second) using Metafluor software (Universal Imaging Corporation).
Random access monochromator
Manufactured by Horiba
Sourced in United States
The Random Access Monochromator is a specialized laboratory instrument used to select specific wavelengths of light from a broader spectrum. It allows for rapid and precise selection of wavelengths, enabling users to conduct highly targeted optical measurements and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Metafluor software, by Universal Imaging (5 mentions)
Fura 2 am, by Thermo Fisher Scientific (4 mentions)
Cascade 512b, by Teledyne (4 mentions)
Hanks balanced salt solution (hbss), by Merck Group (3 mentions)
Te2000 e, by Nikon (3 mentions)
Fura 2 am, by Merck Group (2 mentions)
Ionomycin calcium salt from streptomyces conglobatus, by Merck Group (2 mentions)
Rhod 2 am, by Thermo Fisher Scientific (1 mentions)
5 protocols using random access monochromator
1
Intracellular Calcium Imaging in PASMCs and PAECs
2
Calcium Imaging of B16 Cells
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B16 cells cultured on 35-mm dishes were incubated in culture medium containing 3.5 μmol/L FURA-2-AM (Invitrogen) for 1 h at 37 °C, and then rinsed with Hanks’ Balanced Salt Solution (HBSS, Sigma). Each dish was placed into a culture chamber at 37 °C on the stage of an inverted fluorescence microscope (TE2000E, Nikon Instruments, Fi, Italy), connected to a cooled CCD camera (512B Cascade, Roper Scientific, Ottobrunn, Germany). Samples were illuminated alternately at 340 and 380 nm using a random access monochromator (Photon Technology International, New Jersey, USA) and emission was detected using a 510 nm emission filter. Images were acquired (1 ratio image per s) using Metafluor software (Universal Imaging Corporation, Downington PA, USA). Calibration was obtained at the end of each experiment by maximally increasing intracellular Ca2+-dependent FURA-2AM fluorescence with 5 μmol/L ionomycin (ionomycin calcium salt from Streptomyces conglobatus, Sigma) followed by recording minimal fluorescence in a Ca2+-free medium. [Ca2+]i was calculated according to the formulas previously described54 (link).
3
Measuring Intracellular Calcium Dynamics
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Cells were incubated in EGM-2 containing 3.5 µM Fura-2-AM (Invitrogen) for 1 h at 37 °C and then rinsed with either Hanks’ Balanced Salt Solution (HBSS, Sigma) or Ca2+ free Krebs Henseleit Hepes buffer (KHH). Dishes were placed into a culture chamber kept at 37 °C controlled temperature on the stage of an inverted microfluorimeter (Nikon TE2000E) connected to a cooled CCD camera (512B Cascade, Princeton Instruments). Samples were illuminated alternately at 340 and 380 nm using a random access monochromator (Photon Technology International) and emission was detected using a 510 nm emission filter. Images were acquired (1 ratio image/s) using Metafluor software (Universal Imaging Corporation). Calibration of the signal was obtained at the end of each experiment by maximally increasing intracellular Ca2+-dependent Fura-2 fluorescence ratio (340/380) with 5 µM ionomycin (ionomycin calcium salt from Streptomyces conglobatus, Sigma) followed by recording minimal ratio in Ca2+-free medium. [Ca2+]i was calculated according to previously described formulas55 (link).
4
Intracellular Calcium Measurement using Fura-2-AM
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Cells were incubated in EGM-2 containing 3.5 μM Fura-2-AM (Invitrogen) for 1 h at 37°C and then rinsed with Hanks' Balanced Salt Solution (HBSS Sigma) or Krebs-Henseleit-HEPES (KHH) buffer (140 mM Na+, 5.3 mM K+, 132.4 mM Cl−, 0.98 mM PO42−, 1.25 mM Ca2+, 0.81 mM Mg2+, 5.5 mM glucose, and 20 mM HEPES). Plates were placed into a culture chamber kept at 37°C controlled temperature on the stage of an inverted microfluorimeter (Nikon TE2000E) connected to a cooled CCD camera (512B Cascade, Princeton Instruments). Samples were illuminated alternately at 340 and 380 nm using a random access monochromator (Photon Technology International) and emission was detected using a 510 nm emission filter. Images were acquired (1 image/sec ratio) using MetaFluor software (Universal Imaging Corporation). Calibration of the signal was obtained at the end of each experiment by maximally increasing intracellular Ca2+-dependent Fura-2-AM fluorescence with 5 μM ionomycin (ionomycin calcium salt from Streptomyces conglobatus, Sigma) followed by recording minimal fluorescence in Ca2+-free medium. [Ca2+]i was calculated according to previously described formulas [33 (link)].
5
Mitochondrial Calcium Dynamics in c-FLIP Knockout Cells
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A total of 15 × 104 WT and c-FLIP−/− MEFs were seeded on 35 mm, high, glass bottom Petri dishes. Twenty-four hours after seeding, cells were incubated for 1 h at 37 °C in culture medium containing Rhod2-AM 6 μM (Invitrogen). The ester forms of this calcium indicator are cationic, resulting in potential-driven uptake into mitochondria. Cells were then washed with standard medium and EGTA-containing HBSS at room temperature. Single-cell fluorescence was excited at 545 nm using a random access monochromator (Photon Technology International) and emission was detected using a 580 nm emission filter. Images of the emitted fluorescence, obtained with a × 60 oil objective, were collected using Metafluor software (Universal Imaging Corporation). Where indicated, c-FLIP−/− cells were transiently transfected with pBluescript or c-FLIPL plasmids and mitochondrial Ca2+ entry were measured in Rhod-2AM-loaded cells.
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