Figure 1.
Live cell mitochondrial staining exhibits high mitochondrial reactivity in myotubes but not undifferentiated cells.
C2C12 cells were seeded on the cell culture dishes. Upon reaching confluence, the cells were transferred to DM to induce MT formation. The DM was changed every 2 days. MitoTracker and Hoechst were added into the media for fluorescence staining of mitochondria and nuclei. After 30 min of incubation at 37°C, live-cell images were recorded by bright field or fluorescence microscopy at day2 (A) and day4 (B). The cells were kept for 4days in DM with/without Hoechst 33342 (1 µM), and then Hoechst was added to stain nuclei (C). Cell morphology was also recorded by bright-field phase-contrast microscopy. MitoTracker and Hoechst signals are shown in red and blue, respectively (scale bar in A = 10 µm, B = 100 µm, and C = 50 µm).
Figure 2.
Differentiating cells are distinguishable by MitoTracker reactivity.
C2C12 cells were seeded on the cell culture dishes and induced MT formation as described above. At the indicated day, the cells were double stained by MitoTracker and Hoechst 33342 as described above. Live-cell images were recorded by both bright field phase-contrast and fluorescence microscopy. MitoTracker and Hoechst signals are shown in red and blue respectively (A) (scale bar = 50 µm). Total number of nuclei, number of nuclei in the multi-nucleated (≥2) cells, and in MitoTracker positive cells per each micrograph were counted (n = 6). The average of each categories was graphed (mean ± st.dev) in panel B. (C) The percentage of nuclei in multinucleated (≥2) cells and MitoTracker positive cells over the total number of nuclei per field (n = 6) were calculated and graphed in C (mean ± st.dev) * (P<0.05 from confluent cells). (D) The same numbers of C2C12 cells were seeded on the cell culture dish (94-well plate (Nalge Nunc International)), and differentiation induced accordingly. Red fluorescent signal was measured by a plate reader, and the average values (n = 4) relative to that of the confluent condition (CONF (0)) were graphed (mean ± st.dev).
Figure 3.
MyHC and MyoG positive cells are also MitoTracker positive.
C2C12 cells were seeded on the cell culture dishes, and at the indicated day, live-cell morphology was recorded by a bright field phase-contrast microscopy. The cells were harvested and subjected to (A) western blotting for analysis of expression levels of the indicated proteins. Actin levels show equal total protein loading. (B) The cells kept for the indicated number of days in the DM were double stained with MitoTracker and Hoechst. Expression of MyoG or MyHC was visualized by immune-fluorescence. Representative micrographs shown were recorded by both bright field phase-contrast and fluorescence microscopy. MitoTracker, Hoechst, and MyoG or MyHC signals are shown in red, blue, and green respectively (scale bar in = 50 µm).
Figure 4.
MyHC and MyoG positive cells are also MitoTracker positive.
C2C12 cells were seeded on glass-bottom dishes. Upon reaching confluence, the cells were kept in DM for the indicated number of days to induce MT formation. Cells were subsequently double stained with MitoTracker and Hoechst as described above. Expression of MyoG or MyHC was visualized by immunofluorescence. Representative micrographs shown were recorded by fluorescence microscopy. MitoTracker, Hoechst, and MyoG or MyHC signals are shown in red, blue, and green respectively (scale bar in = 10 µm).
Table 1.
Quantification of correlation between MyoG or MyHC positive cells and MitoTracker positive cells.
Figure 5.
MyoD-converted myocytes from fibroblasts can be identified by MitoTracker.
MyoD expression vector (pMT2-MyoD) or empty vector (pMT2) with either pCMV-EGFP or pMCK-EGFP was co-transfected into C3H10T1/2 fibroblasts. The cells were allowed an initial 2 days recovery in GM, followed by DM for 2 days. Nuclei and mitochondria were live-stained with Hoechst and MitoTracker and EGFP signal was recorded by fluorescence microscopy (scale bar in A = 50 µm, B = 10 µm).
Figure 6.
Mitochondrial activity responds to a positive or a negative regulator of myogenesis.
C2C12 cells were transfected with p53-EGFP, EYFP-JDP2, or EYFP-NLS. Expression of fusion proteins, mitochondria (MitoTracker), and nuclei (Hoechst 33342) were visualized by fluorescence microscopy as described in Methods section. Micrographs were taken at 24 hrs after transfection (cells were in GM for 24 hars) in A, or 72 hrs after transfection (cells were kept in 48 hrs in DM) in B. MitoTracker, Hoechst, and transfected fusion proteins signals are shown in red, blue, and green respectively. White arrow indicates cells expressing an indicated fluorescence fusion protein. Merged image is shown in the middle panel. Intensity of MitoTracker signals were measured using ImageJ program (NIH) (top panel) based on the MitoTracker signal (Bottom panel). Micrographs shown are representative of the samples (n≥6) (scale bar = 10 µm).