Visualization of MMP loss was performed by JC-1 staining. A2780s and A2780cp cells were seeded on confocal disc, treated with Ssd and/or CDDP and stained with JC-1 dye (10 μg/ml, 30 min, 37°C). The retained MMP (Tetramethylrhodamine-isothiocyanate (TRITC) signal) and the loss (Fluoresceinisothiocyanate (FITC) signal) were real-time examined and captured under epifluorescence microscopy (60X objective; Applied Precision DeltaVision Elite, Applied Precision, Inc, USA). At least 500 cells were analyzed per experimental group.
Applied precision deltavision elite
Manufactured by Cytiva
Sourced in United States
The Applied Precision DeltaVision Elite is a high-performance, widefield fluorescence microscope system designed for advanced live-cell and fixed-sample imaging. It provides exceptional optical performance and flexibility to support a wide range of applications in life science research.
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7 protocols using applied precision deltavision elite
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Mitochondrial Membrane Potential Visualization
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Lysosome Calcium Measurement with GCaMP3-ML1
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Lysosome calcium measurement was performed using methods described previously [10 (link)]. Briefly, 2 × 105 HT1080 cells stably expressing GCaMP3-ML1 were cultured in a 35-mm confocal dish (SPL Life Sciences, 100350). Changes in cytosolic Ca2+ levels were monitored by following changes in GCaMP3-ML1 fluorescence for 15 min upon addition of 200 μM GPN in Ca2+-free external solution containing 145 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM glucose, 1 mM EGTA (Sigma-Aldrich, E3889), 20 mM HEPES (Gibco, 15630080), pH 7.4, using the real-time mode of epifluorescence microscopy (Applied Precision DeltaVision Elite, Applied Precision Inc., USA). Data Inspection Program provided by the DeltaVision software was used to measure the intensity of GCaMP3-ML1 fluorescence, and the mean fluorescence intensity was monitored at 488 nm. The acquired epifluorescence images were numerically deconvolved using DeltaVision algorithms (Applied Precision Inc., USA).
3
Peroxynitrite Production Evaluation Using DHR
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DHR oxidation was used to evaluate peroxynitrite production of cells exposed, as previously described [13 (link)]. Cells were grown in a confocal dish, and liver paraffin tissue was replaced with 0.5 mL of Dulbecco’s phosphate-buffered solution (dPBS) containing 20 μM DHR (Invitrogen, Carlsbad, CA, USA) at 37 °C for 30 min. The detection of rhodamine 123 (ex 485 nm/em 520 nm), an oxidation product of DHR, after exposure to the different experimental conditions was followed online in epifluorescence (Applied Precision Delta Vision Elite, Applied Precision Inc., Issaquah, WA, USA) with the subtraction of background fluorescence.
4
Monitoring Cytosolic Calcium Dynamics in HeLa Cells
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2 × 105 HeLa cells were cultured in 35 mm confocal disk at 37°C CO2 incubator for 24 h. 5 mM of Fluo 3/AM/DMSO stock solution was diluted to 5 μM working solution using Hanks-balanced salt solution (HBSS) and then added to cells at 37°C for 30 min. HeLa cells were then washed three times with HEPES buffer saline and incubated at 37°C in an imaging chamber for another 10 min. Changes in cytosolic [Ca2+] levels were monitored by following changes in fluo-3 fluorescence upon addition of 10 μM LP-4 in HBSS buffer, using the real-time mode for 5 min by epifluorescence microscopy (Applied Precision DeltaVision Elite, Applied Precision, Inc., United States). Data Inspection Program provided by the DeltaVision software was used to measure the intensity of the fluo-3 fluorescence and the mean fluorescence intensity was monitored at 523 nm and plotted against time (sec).
5
Monitoring Calcium Signaling in RASFs
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2 × 105 RASFs cells were cultured in 35 mm confocal disc at 37°C CO2 incubator for 24 h. FLIPR Calcium 6 reagent was added to cells at 37°C for 30 min. RASFs were then washed 3 times with HEPES buffer saline and incubated at 37°C in an imaging chamber for another 10 min. Changes in cytosolic [Ca2+] levels were monitored by following changes in FLIPR Calcium 6 fluorescence upon addition of 1 μM celastrol in HBSS buffer, using the real-time mode for 4 min by epifluorescence microscopy (Applied Precision DeltaVision Elite, Applied Precision, Inc., USA). Data Inspection Program provided by the DeltaVision software was used to measure the intensity of the FLIPR Calcium 6 fluorescence and the mean fluorescence intensity was monitored at 525 nm and plotted against time (s).
6
Peroxynitrite Production Evaluation
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DHR oxidation was used to evaluate peroxynitrite production of cells exposed, as previously described (Munhoz et al., 2012 ). Cells and aortic ring were replaced with Dulbecco's phosphate‐buffered saline (dPBS) containing 20 µM DHR (Invitrogen, Carlsbad, CA, USA) at 37°C for 30 min. The oxidation product of DHR was detected after exposure to different experimental conditions in epifluorescence (Applied Precision Delta Vision Elite, Applied Precision Inc., Issaquah, WA, USA).
7
Fluo-3 Calcium Imaging in HeLa Cells
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2 × 105 HeLa cells were cultured in 35 mm confocal disc at 37 °C CO2 incubator for 24 h. 5 mM of Fluo 3/AM/DMSO stock solution was diluted to 5 μM working solution using Ca2+ free Hanks-balanced salt solution (HBSS) and then added to cells at 37 °C for 30 min. HeLa cells were then washed 3 times with HEPES buffer saline and incubated at 37 °C in an imaging chamber for another 10 min. Changes in cytosolic [Ca2+] levels were monitored by following changes in fluo-3 fluorescence upon addition of 10 μM neferine in Ca2+ free HBSS buffer, using the real-time mode for 5 minutes by epifluorescence microscopy (Applied Precision DeltaVision Elite, Applied Precision, Inc, USA). Data Inspection Program provided by the DeltaVision software was used to measure the intensity of the fluo-3 fluorescence and the mean fluorescence intensity was monitored at 523 nm and plotted against time (sec).
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