PC12 cells were transfected twice with siRNA and with VMAT2-pHluorin, as well on day 1 before imaging, as previously described (Asensio et al., 2010 (link); Onoa et al., 2010 (link)). Glass coverslips containing the transfected PC12 cells were mounted in the perfusion chamber (Warner Instruments) of an inverted Nikon TE3000 microscope fitted with a 60× water objective, and the cells were imaged in modified Tyrode’s solution at room temperature with stimulation by 90 mM K+ and alkalinization of the quenched intracellular fluorophore with 10 mM NH4Cl. Cells were illuminated using a Xenon lamp (Sutter Instruments) with 470/40-nm excitation and 525/50-nm emission filters. Images were acquired every second using a QuantEM charge coupled device camera (Photometrics). MetaMorph software was used to control data collection and perform analysis offline.
Perfusion chamber
Manufactured by Warner Instruments
Sourced in United States
The Perfusion Chamber is a laboratory equipment designed to facilitate the study of live cells in a controlled environment. Its core function is to provide a closed and regulated chamber that allows for the continuous delivery and exchange of culture media, enabling researchers to observe and analyze cellular behavior and responses under various experimental conditions.
Automatically generated - may contain errors
Lab products found in correlation
Te3000 microscope, by Nikon (1 mentions)
Xenon lamp, by Sutter Instruments (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Stage top incubator, by Okolab (1 mentions)
Imaris analysis software, by Oxford Instruments (1 mentions)
Ti e microscope, by Yokogawa (1 mentions)
Hq2 cooled ccd camera, by Teledyne (1 mentions)
Csu x confocal scanhead, by Yokogawa (1 mentions)
Axiovert 200 inverted microscope, by Zeiss (1 mentions)
Metafluor software, by Molecular Devices (1 mentions)
7 protocols using perfusion chamber
1
Live Imaging of Synaptic Vesicle Dynamics
2
Imaging of Transferrin Receptor Dynamics
3
Monitoring Cytosolic Ca2+ Signaling in HEK293 Cells
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Cytosolic Ca2+ levels in stable, inducible HEK293 cells expressing RyR2 WT or the H29D mutant were monitored using single-cell Ca2+ imaging and the fluorescent Ca2+ indicator dye Fura-2 as described previously [18 (link),26 (link),31 (link)]. Briefly, cells grown on glass coverslips for 8-18h after induction (as indicated) by 1 μg/ml tetracycline (Sigma) were loaded with 5 μM Fura-2, AM in KRH buffer (125 mM NaCl, 5 mM KCl, 6 mM glucose, 1.2 mM MgCl2 and 25 mM Hepes, pH 7.4) plus 0.02% pluronic F-127 and 0.1 mg/ml BSA for 20 min at room temperature (23°C). The coverslips were then mounted in a perfusion chamber (Warner Instruments) on an inverted microscope (Nikon TE2000-S). The cells were perfused continuously with KRH buffer containing increasing extracellular Ca2+ concentrations (0, 0.1, 0.2, 0.3, 0.5, 1.0 and 2.0 mM). Caffeine (10 mM) was applied at the end of each experiment to confirm the expression of active RyR2 channels. Time-lapse images (0.25 frame/s) were captured and analyzed with Compix Simple PCI 6 software. Fluorescence intensities were measured from regions of interest centered on individual cells. Only cells that responded to caffeine were analyzed. The filters used for Fura-2 imaging were λex = 340±26 nm and 387±11 nm, and λem = 510±84 nm with a dichroic mirror (410 nM).
4
Live Cell Imaging of Rho Kinase Inhibition
5
Live Cell Traction Force Microscopy
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Live cell traction force measurements were performed on an inverted Nikon Ti-E microscope with a CSU-X confocal scanhead (Yokogawa), laser merge module containing 491, 561 and 642 nm laser lines (Spectral Applied Research) and an HQ2 cooled CCD camera (Roper Scientific). All hardware was controlled via Metamorph acquisition software (MDS Analytical Technologies). Traction force data was obtained at 37°C in a perfusion chamber (Warner Instruments) using a 60x 1.2 NA Plan Apo WI objective (Nikon). Cells were maintained in culture media supplemented with 10 mM HEPES and 30 μl/ml Oxyrase (Oxyrase, Inc.).
6
Measuring H2O2 Permeability in Aquaporin
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In order to test aquaporin for H2O2 channeling, 30,000 cells were seeded on a 15 mm coverslip in 100 µL, and left for 24 h to attach. The next day, cells were treated with 2.5 mM MBCD for 2 h. After the MBCD treatment, cells were treated with 0.1 µg/mL EGF for 24 h. Before measuring the H2O2 permeability, cells were incubated with 10 µM H2-DCFDA for 30 min at 37 °C and 5% CO2. Next, the coverslips were washed with 25 mM HEPES and mounted in a perfusion chamber (Warner Instruments, Hamden, CT, USA) on a Zeiss Axiovert 200 inverted microscope. Fluorescence was excited at a wavelength of 495/10 nm using the Metafluor Software (Molecular Devices, Sunnyvale, CA, USA), which was used for data recording. Cells were equilibrated in 25 mM HEPES for 1 min, after which 100 µM H2O2 was added to cells, and fluorescence was measured every 10 s. H2O2 intake was obtained from the slope of a plot of fluorescence intensity over time and analyzed using Microsoft Excel Visual Basic Analysis code (https://github.com/nijelic/slope-residuals-for-multivariate-time-series , accessed on 19 January 2023).
7
Imaging of Transferrin Receptor Dynamics
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Imaging of super-ecliptic pHluorin-tagged transferrin receptor was
conducted essentially as previously described (Keith et al., 2012 (link); Woolfrey et al., 2015 (link)). DIV 11–14
hippocampal neurons from WT and AKAP150CS mouse cultures were transfected
(Lipofectamine 2000) with plasmids encoding SEP-TfR and mCherry (as a cell
fill) and imaged 3 days later. Imaging was conducted on the spinning-disk
confocal microscope detailed above. Prior to imaging, neurons were incubated
in ACSF plus 1 mM MgCl2 for 30 min and were maintained during
imaging at 33–35°C in a perfusion chamber (Warner
Instruments). Baseline rates of SEP-TfR exocytic events (events defined as
2.5-fold above the median intensity of the dendrite) were determined by
acquiring z stacks of 10 optical sections (1.0 μm spacing) every 6 s
for 5 min.
conducted essentially as previously described (Keith et al., 2012 (link); Woolfrey et al., 2015 (link)). DIV 11–14
hippocampal neurons from WT and AKAP150CS mouse cultures were transfected
(Lipofectamine 2000) with plasmids encoding SEP-TfR and mCherry (as a cell
fill) and imaged 3 days later. Imaging was conducted on the spinning-disk
confocal microscope detailed above. Prior to imaging, neurons were incubated
in ACSF plus 1 mM MgCl2 for 30 min and were maintained during
imaging at 33–35°C in a perfusion chamber (Warner
Instruments). Baseline rates of SEP-TfR exocytic events (events defined as
2.5-fold above the median intensity of the dendrite) were determined by
acquiring z stacks of 10 optical sections (1.0 μm spacing) every 6 s
for 5 min.
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