For ROS generation measurements, hippocampal neurons were exposed to either vehicle or Aβo 1 μM for 24 h. Then, cells were washed in SEM and loaded with the ROS probe 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA 2 μM) for 40 min at RT in the dark. Then coverslips containing cells were placed on a Zeiss Axiovert 100 TV inverted microscope and subjected to fluorescence imaging. Fluorescence images were captured with the FITC filter set with a Hamamatsu ER-Orca fluorescence camera.
Axiovert 100 tv inverted microscope
Manufactured by Zeiss
Sourced in Israel
The Axiovert 100 TV is an inverted microscope designed for observation and analysis in the laboratory. It features a stable stand, versatile illumination options, and high-quality optics to provide clear and detailed images of specimens. The core function of this microscope is to enable researchers and technicians to observe and study samples effectively in a wide range of applications.
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6 protocols using axiovert 100 tv inverted microscope
1
Measuring Aβ-Induced ROS in Hippocampal Neurons
2
Assessing Cell Migration in Fibroblasts
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GFP/Fb and bcat-GFP/Fb were treated with 500 ng/ml doxycycline for 48 h and subsequently seeded in 24-well tissue culture plates in triplicate at a density of 80,000 cells/well in DMEM supplemented with 10% FBS and 1% PS. Fibroblasts were incubated in a humidified incubator at 37 °C with 5% CO2 for one day to allow them to attach and reach 90% confluence. Culture medium was then switched to serum-reduced medium (DMEM supplemented with 0.5% FBS or SK-MEL-24-conditioned DMEM medium supplemented with 0.5% FBS) supplemented with 0.02 mg/ml mitomycin C (Sigma, St. Louis, MO), which inhibited cell proliferation during the procedure. After two days, mitomycin C was removed, and the fibroblast layer was scratched using a p200 pipet tip in the center of each well to form a rectangular cell-free area. Fibroblasts were allowed to migrate for 24–48 h depending on the experimental design. Bright-field images were captured using a Carl Zeiss Axiovert 100 TV inverted microscope at the indicated time points.
3
Mitochondrial Potential Imaging in Hippocampal Neurons
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For mitochondrial membrane potential measurements, hippocampal neurons were exposed to either vehicle or Aβo 1 μM for 24 h. Then, cells were washed in SEM and loaded with the mitochondrial potential probe (TMRM, 10 nM) for 30 min at RT in the dark. Then coverslips containing cells were placed on a Zeiss Axiovert 100 TV inverted microscope and subjected to fluorescence imaging. Fluorescence images were captured with the rhodamine filter set with a Hamamatsu ER-Orca fluorescence camera as reported previously (Calvo-Rodríguez et al., 2016b (link)).
4
Intracellular Ca2+ Signaling in hiPSC-CMs
5
Mitochondrial Membrane Potential Measurement
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Cells were loaded with TMRM (10 nM, 30 min) at room temperature and placed on a Zeiss Axiovert 100 TV inverted microscope. For comparison of ΔΨ in NCM460 and HT29 cells, cells were loaded in parallel and TMRM fluorescence images were captured using exactly the same imaging parameters. Then, cells were treated with FCCP 10 μM to collapse mitochondrial potential. After 10 min, a fluorescence image was taken to be used as background image. For monitoring effects of NSAIDs on ΔΨ, TRMR-loaded cells were subjected to fluorescence imaging at 5 s intervals. At the end of the experiment, cells were treated with FCCP 10 μM for 10 min before capturing a fluorescence image to be used as background fluorescence for cells. Fluorescence intensity from regions of interest corresponding to individual cells were average and the average value after collapse of the mitochondria potential with FCCP was substracted from each region of interest. Fluorescence values from individual cells were normalized (expressed as the percentage value of fluorescence before addition of NSAIDs) and averaged. To quantify mitochondrial depolarization, the effect of NSAIDs or FCCP on the ratio of TMRM fluorescence in mitochondria relative to that surrounding cytosol was calculated as reported previously [17 (link), 18 (link)].
6
Imaging Mitochondrial Potential in Hippocampal Neurons
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Hippocampal neurons were washed in SEM and loaded with the mitochondrial potential probe tetramethylrhodamine, methyl ester (TMRM, 10 nM) for 30 min at room temperature in the dark. Then coverslips containing cells were placed on a Zeiss Axiovert 100 TV inverted microscope and subjected to fluorescence imaging. Fluorescence images were captured with the rhodamine filter set with a Hamamatsu ER-Orca fluorescence camera as reported previously [4, 14, 31] .
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