The mitochondrial membrane potential (ΔΨm) was measured using the mitochondrial membrane potential—specific fluorescent probe, JC-1 (Molecular Probes). H9c2 cells were seeded on glass coverslips and cultured at least overnight before the experiment. After treatment, cells were washed twice in ice-cold PBS and then incubated for 5 min at 37°C in modified Krebs-Henseleit solution (118mM NaCl, 5mM KCl, 1.2 mM KH2PO4, 25 mM NaHCO3, 5mM glucose, 1.2 mM MgSO4x7H2O) containing 0.1 μM JC-1. When excited at 488 nm, the dye emits green fluorescence (530 nm) at low ΔΨm and red (590 nm) at high ΔΨm. Following incubation, the cells were washed once with Krebs-Henseleit solution and then imaged with a Nikon Eclipse Ti-U fluorescent microscope equipped with a Spot RT3 camera using a 60x objective and epifluorescent illumination. The investigator collecting data was blinded to treatment groups. All experiments were repeated in triplicate.
Spot rt3
Manufactured by Nikon
The Spot RT3 is a digital camera system designed for microscopy applications. It features a high-resolution CCD sensor and advanced image processing capabilities to capture detailed, high-quality images. The Spot RT3 is compatible with a wide range of microscopes and can be used in various scientific and research settings.
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Lab products found in correlation
7 protocols using spot rt3
1
Measuring Mitochondrial Membrane Potential
2
Muscle Fiber Cross-Sectional Area Analysis
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H&E-stained muscle cross-sections were digitally captured on the Nikon Eclipse E600 with the Spot RT3 digital camera. Choosing multiple areas enriched with muscle fibers with centrally localized myonuclei, the circumference of each fiber was outlined using Image Studio Lite (LI-COR) to generate fiber cross-sectional area (CSA). More than 300 fibers were analyzed for each mouse. Microsoft Excel was used to process the CSA data and to generate graphs. Adobe Illustrator was used to format the graphs.
3
In Situ Hybridization of Prg4 in Tissues
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In situ hybridization was performed on frozen or paraffin sections using digoxigenin (DIG)-labeled probes and color detected with BM purple (Roche, IN, USA), as previously described [21] (link), [22] (link). The Prg4 mouse probe used was generated by PCR (nucleotides 2370–3070). Images were captured on a Nikon Eclipse 80i microscope with a Spot RT3 camera.
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In situ hybridization was performed on frozen or paraffin sections using digoxigenin (DIG)-labeled probes and color detected with BM purple (Roche, IN, USA), as previously described [21] (link), [22] (link). The Prg4 mouse probe used was generated by PCR (nucleotides 2370–3070). Images were captured on a Nikon Eclipse 80i microscope with a Spot RT3 camera.
4
Apoptosis Quantification via Hoechst Staining
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2,000 cells/well were seeded in 96-well plates and cultured overnight before treatments. Following treatments, the cells were washed with 1 × PBS and incubated with 0.5 μg/ml Hoechst 33342 for 10 min. Nuclei were visualized by a Nikon Eclipse Ti-U fluorescence microscope equipped with a Spot RT3 camera using 4× and 20× objective lenses. Images were recorded using a 4× objective lens. Nuclei having condensed or fragmented apoptotic characteristics were quantified using the ImageJ software (NIH). For each treatment, at least 300 nuclei were evaluated.
5
Lung Tissue Histology Protocol
6
Measurement of Mitochondrial Membrane Potential
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The mitochondrial membrane potential (ΔΨm) was measured using the mitochondrial membrane potential specific fluorescent probe, JC-1 (Enzo Life Sciences, ENZ-52304) as we performed it earlier [25 (link)]. NRCM cells were seeded on glass coverslips coated with gelatin and cultured for at least 2 days before the experiment. After the treatment, cells were washed in PBS and incubated for 15 min at 37°C in media containing 5 μg/mL JC-1. When excited at 488 nm, the dye emits red fluorescence (590 nm) at high ΔΨm and green (530 nm) at low ΔΨm. Following incubation, the cells were washed once with PBS and then imaged with a Nikon Eclipse Ti-U fluorescent microscope equipped with a Spot RT3 camera using a 60x objective and epifluorescent illumination. All experiments were repeated three times. Fluorescent signals were quantified by using the ImageJ software (NIH, Bethesda, MD, USA).
7
Myogenic Differentiation Kinetics
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