All fluorescent microscopy imaging experiments were performed on an inverted Olympus IX81 microscope equipped with a Hamamatsu C8484 camera, 60X oil immersion Plan S-Apo objective and CFP, TxRed, and Cy5.5 filter cubes from Semrock. Images were taken at 250 ms exposure for CFP fluorescence, 500 ms exposure for mCh fluorescence, and 1000 ms exposure for Alexa 647 fluorescence. An ROI surrounding the entire cell was defined for cells that contain both the PKA-Reporter and Cry2-mCh. The minimum background fluorescence for each image was subtracted from each cell and then the ratio of the phosphoPKA-Reporter fluorescence/PKA-Reporter fluorescence (Alexa 647/CFP) was calculated. Values were normalized to the ratio from cells that were never exposed to light. All imaging analysis was done using Image J software and reported as the mean ± standard error of 10 – 25 cells per experiment.
C8484 camera
Manufactured by Hamamatsu Photonics
The C8484 is a high-performance CCD camera developed by Hamamatsu Photonics. It features a low-noise, high-resolution CCD image sensor and offers a range of technical specifications suitable for various scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Ix71 microscope, by Olympus (5 mentions)
Poly d lysine, by Merck Group (5 mentions)
Rat tail collagen 1, by Thermo Fisher Scientific (3 mentions)
Hci image software, by Hamamatsu Photonics (3 mentions)
Glass bottom dishes, by MatTek (2 mentions)
603 oil objective, by Olympus (2 mentions)
Hcimage software, by Hamamatsu Photonics (2 mentions)
Ix81 microscope, by Hamamatsu Photonics (1 mentions)
Glass bottom dishes, by Merck Group (1 mentions)
6 protocols using c8484 camera
1
Quantifying Light-Induced PKA Activation
2
Quantifying Mitochondrial Morphology Changes
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HeLa cells transfected with mtGFP or MEFmtGFP cells were seeded at a density of 100,000 cells/well on glass‐bottom dishes (MatTek) coated with poly‐D‐lysine (Sigma) or rat tail collagen 1 (GIBCO). Cells were then treated as indicated and images were recorded with an Olympus IX71 microscope with 60× oil objective (Olympus), a Hamamatsu C8484 camera (Hamamatsu Photonics), and HCI image software (Hamamatsu Photonics). Quantification was performed by blinding the images and then scoring cells based on the presence of primarily fragmented, tubular, or elongated mitochondria, as before (Lebeau et al, 2018 (link)). At least three different researchers scored each set of images and these scores were averaged for each individual experiment and all quantifications shown were performed for at least three independent experiments quantifying a total of > 60 cells/condition across all experiments. The data were then prepared in PRISM (GraphPad, San Diego, CA) and plotted on a stacked bar plot to show the average morphology and standard error of the mean across all experiments. Statistical comparisons were performed using a two‐way ANOVA in PRISM, comparing the relative amounts of fragmented, tubular, or elongated mitochondria across different conditions.
3
Mitochondrial Morphology Quantification
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MEF or HeLa cells transiently transfected with mtGFP or MEFmtGFP cells were seeded at a density of 100,000 cells/well on glass-bottom dishes (MatTek) coated with poly-D-lysine (Sigma) or rat tail collagen 1 (GIBCO). Cells were then treated as indicated and images were recorded with an Olympus IX71 microscope with 60x oil objective (Olympus), a Hamamatsu C8484 camera (Hamamatsu Photonics), and HCI image software (Hamamatsu Photonics). Quantification was performed by blinding the images and then scoring cells based on the presence of primarily fragmented, tubular, or elongated mitochondria, as before (Lebeau et al., 2018 (link)). At least three different researchers scored each set of images and these scores were averaged for each individual experiment and all quantifications shown were performed for at least 3 independent experiments quantifying a total of >60 cells/condition across all experiments. The data were then analyzed in PRISM (GraphPad, San Diego, CA) and plotted on a stacked bar plot to show the average morphology and standard error of the mean across all experiments. Statistical comparisons were performed using a 2-way ANOVA in PRISM, comparing the relative amounts of fragmented, tubular, or elongated mitochondria across different conditions.
4
Fluorescence Microscopy of Mitochondrial Morphology
5
Fluorescence Microscopy of Mitochondrial Morphology
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Cells were seeded the day before on glass-bottom dishes coated with poly-D-lysine (Sigma) to reach a confluence of 50% the day of the experiment. Cells were treated in media with reagents for the indicated time and then washed 3 times with HBSS containing 10% FBS, 100 U/mL−1 penicillin, and 100 μg/mL−1 streptomycin and put in the same buffer for imaging. Images were then recorded with an Olympus IX71 microscope with 603 oil objective (Olympus), a Hamamatsu C8484 camera (Hamamatsu Photonics), and HCImage software (Hamamatsu Photonics). For quantification, we collected images for more than 30 cells per condition and then blinded these images. Three researchers then scored the cells for containing primarily fragmented, tubular, or elongated mitochondria (see Figure S1A for examples of scoring). The scores from the three researchers were then averaged and combined with other independent experiments in the data presented in the bar graphs shown throughout the paper.
6
Mitochondrial Morphology Quantification
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HeLa cells transfected with mt GFP or MEF mtGFP cells were seeded at a density of 100,000 cells/well on glassbottom dishes (MatTek) coated with poly-D-lysine (Sigma) or rat tail collagen 1 (GIBCO). Cells were then treated as indicated and images were recorded with an Olympus IX71 microscope with 60x oil objective (Olympus), a Hamamatsu C8484 camera (Hamamatsu Photonics), and HCI image software (Hamamatsu Photonics). At least 20 cells were imaged per condition for each experiment for quantification. Quantification was performed by blinding the images and then scoring cells based on the presence of primarily fragmented, tubular, or elongated mitochondria, as before 38 (link) . Three different researchers scored each set of images and these scores were averaged for each individual experiment. All quantifications shown were performed for at least 3 independent experiments, where averages in morphology quantified from each individual experiment were then combined. The data were then prepared in PRISM (GraphPad, San Diego, CA) and plotted on a stacked bar plot to show the average morphology and standard error of the mean across all experiments.
Statistical comparisons were performed using a 2-way ANOVA in PRISM, comparing the relative amounts of fragmented, tubular, or elongated mitochondria across different conditions.
Statistical comparisons were performed using a 2-way ANOVA in PRISM, comparing the relative amounts of fragmented, tubular, or elongated mitochondria across different conditions.
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