Intracellular Ca2+ measurements were obtained from Fura-2 (Invitrogen) AM-loaded myotubes as described previously (Goonasekera et al., 2007 (link)). Briefly, myotubes were differentiated for 5–6 days on glass bottom dishes and loaded with 5 μM Fura-2 AM for 45 min at 37°C in a normal rodent Ringer’s solution consisting of 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4. Coverslips of Fura-2–loaded cells were then mounted in a tissue chamber on the stage of an epifluorescence-equipped inverted microscope (Zeiss). Cells were sequentially excited at 340- and 380 nm wavelength and fluorescence emission at 510 nm was collected using a high-speed CCD camera (Hamamatsu). The results are presented as the ratio of 340/380 nm. Maximal increase or peak change in intracellular Ca2+ by induction of 10 mM caffeine was defined as the difference between peak and 10 s of baseline fluorescence ratios prior to addition of caffeine. To better visualize differences in peak change across multiple treatment groups, 340/380 ratios for each individual myofiber were normalized to their own 10 s of baseline 340/380 ratios prior to addition of caffeine. Data are also presented without normalization in Figure 5—figure supplement 1D–F .
Epifluorescence equipped inverted microscope
Manufactured by Zeiss
The Epifluorescence-equipped inverted microscope is a laboratory instrument designed for fluorescence microscopy. It features an illumination system that excites fluorescent dyes or proteins within a sample, allowing for the visualization and analysis of specific cellular structures or molecular interactions.
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Lab products found in correlation
High speed ccd camera, by Hamamatsu Photonics (2 mentions)
Fura 2, by Thermo Fisher Scientific (1 mentions)
Fura 2, by Zeiss (1 mentions)
Lsm800 confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Proteintech (1 mentions)
Goat anti mouse igg h l, by Proteintech (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Fura 2 am, by Zeiss (1 mentions)
3 protocols using epifluorescence equipped inverted microscope
1
Intracellular Calcium Imaging of Myotubes
2
Quantification of DNA Damage and Cardiac Troponin I
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Cells seeded on coverslips were fixed in 3.7% formaldehyde in PBS for 15 minutes at indicated time points. Then cells were washed with PBS and permeabilized for 10 minutes in 0.1% TritonX‐100/PBS. After permeabilization, blocking buffer (5% bovine serum albumin in 0.1% Triton/PBS) was added for 1 hour, followed by incubation with γ‐H2AX (phospho‐histone H2A.X, rabbit recombinant, Cell Signaling, ref 7631S) overnight at 4 °C. After being washed with PBS, cells were incubated with Alexa 594 antirabbit fluorescent secondary antibodies (Proteintech, 1:200 dilution) in the dark for 2 hours at room temperature. The nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, Beyotime, C1002). Images were acquired using Zeiss LSM800 confocal microscope and foci of phospho‐H2AX were quantified using ImageJ software.
For cardiac troponin I staining, sections were blocked with PBS containing 10% fetal bovine serum for 1 hour and then stained overnight at 4 °C in 0.5% BSA with 10 μg/mL of anticardiac troponin I antibody (#66376‐1‐Ig, Proteintech, 1:200 dilution). Secondary staining was performed with 10 μg/mL of goat antimouse IgG (H+L) (#SA00007‐1, Proteintech) for 30 minutes and then subsequently stained with DAPI. Images were collected on a Carl Zeiss epifluorescence‐equipped inverted microscope.
For cardiac troponin I staining, sections were blocked with PBS containing 10% fetal bovine serum for 1 hour and then stained overnight at 4 °C in 0.5% BSA with 10 μg/mL of anticardiac troponin I antibody (#66376‐1‐Ig, Proteintech, 1:200 dilution). Secondary staining was performed with 10 μg/mL of goat antimouse IgG (H+L) (#SA00007‐1, Proteintech) for 30 minutes and then subsequently stained with DAPI. Images were collected on a Carl Zeiss epifluorescence‐equipped inverted microscope.
3
Intracellular Calcium Measurement in Myotubes
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Intracellular calcium [Ca2+]i was measured in day-6 myotubes as previously described64 . Myotubes on coverslips were loaded with 5 mM Fura-2 AM (Invitrogen) in 1x regular rodent Ringer’s solution (RRS) at RT for 45 min. For measurement in heat and caffeine status, myotubes were incubated with 50 μM 4CmC at 37 °C for 10 min just after loaded Fura-2 AM, and then mounted in a chamber set at 37 °C on the stage of an epifluorescence-equipped inverted microscope (Zeiss). Myotubes were sequentially excited at 340 and 380 nm wavelength and fluorescence emission at 510 nm was collected using a high-speed CCD camera (Hamamatsu). The ratio of the fluorescence intensity (R340/380) was analyzed using Volocity Software (PerkinElmer). For calculating the intracellular calcium concentration, the maximum ratio (Rmax) and minimum ratio (Rmin) were determined by using ionomycin (20 μM) in RRS followed by EGTA solution (125 mM) at the end of each experiment. [Ca2+]i of each myotube was calculated as previously described65 (link).
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