Shortly before the experiments, we dissociated cells (i.e., GH3, R1220 or mHippoE-14 cells) and transferred an aliquot of cell suspension to a custom-made recording chamber mounted on the stage of DM-IL inverted fluorescence microscope (Leica; Major Instruments, Kaohsiung, Taiwan). We bathed cells at room temperature (20–25 °C) in HEPES-buffered normal Tyrode’s solution, the composition of which is stated above. The electrodes were prepared from Kimax-51 capillaries (#34500 [1.5–18 mm in outer diameter]; Kimble, Dogger, New Taipei City, Taiwan) by either a P-97 Flaming/Brown puller (Sutter, Novato, CA, USA) or a Narishige PP-830 puller (Narishige; Major Instruments, New Taipei City, Taiwan), and we then fire-polished their tips with MF-83 microforge (Narishige). As filled with different internal solutions, their resistances ranged from 3 to 5 MΩ. Recordings of membrane potential or ionic currents were measured in the whole-cell or cell-attached configuration of the standard patch-clamp technique with an RK-400 patch amplifier (Bio-Logic, Claix, France) [68 (link)]. The liquid junction potentials were zeroed shortly before giga-seal formation was made, and the whole-cell data were corrected.
Dm il inverted fluorescence microscope
Manufactured by Leica camera
The Leica DM IL inverted fluorescence microscope is a laboratory equipment designed for high-performance fluorescence imaging. It features an inverted optical design, allowing for easy sample handling and observation of cells and tissue cultures. The DM IL provides reliable and consistent illumination for fluorescence applications, enabling users to capture detailed images of labeled specimens.
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Lab products found in correlation
P 97 flaming brown puller, by Sutter Instruments (1 mentions)
Rk 400 patch amplifier, by Bio-Logic (1 mentions)
Mf 83 microforge, by Narishige (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 spectral confocal microscope, by Leica camera (1 mentions)
Bodipy 493 503, by Cayman Chemical (1 mentions)
Everbrite mounting medium with 4 6 diamidino 2 phenylindole dapi, by Biotium (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
3 protocols using dm il inverted fluorescence microscope
1
Patch-clamp Electrophysiology of Dissociated Cells
2
Immunofluorescence Staining of ELT-3 Cells
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ELT-3 cells were cultured on sterile glass coverslips and treated with 5 μM Manz A or DMSO for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min followed by permeabilization with 0.1% Triton X-100/PBS (v/v) for 10 min. Cells were then blocked in BlockPRO blocking buffer (Visual Protein, Taipei, Taiwan) at RT for 1 h and incubated with primary antibodies overnight at 4 °C. After being washed three times with Tris-buffered saline containing 0.1% Tween-20/TBS (v/v, TBST), cells were further incubated with secondary antibodies (Alexa Fluor® 488 goat anti-rabbit IgG and Alexa Fluor® 546 goat anti-mouse IgG, Thermo Fisher Scientific) for 1 h at RT in the dark. After several washes with TBST, cells were mounted with EverBrite™ Mounting Medium with DAPI. Fluorescent images were taken with a Leica DM IL inverted fluorescence microscope or a Leica TCS SP5 confocal spectral microscope.
3
Mitochondrial Morphology Analysis
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Cells were seeded on coverslips and treated with 5 μM Manz A or DMSO for 24 h. Cells were stained with 100 nM of MitoTracker™ Red CMXRos (Thermo Fisher Scientific) for 45 min in the dark. Following three washes of PBS, cells were fixed with 4% paraformaldehyde/PBS (w/v) for 15 min and stained with Bodipy 493/503 (Cayman Chemical) for 30 min at RT in the dark. EverBrite™ Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI; Biotium, Fremont, CA, USA) was used to immobilize the coverslips. Coverslips were sealed with nail polish. Fluorescent images were captured using a Leica DM IL inverted fluorescence microscope.
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