4 6 diamidino 2 phenyindole dapi

EP, LP, undifferentiated, differentiated, OTA-, or t-BHQ-treated MSCs were seeded at 5000 cells/cm² on 4-well glass chamber slides (Nalge Nunc International, Rochester, NY, USA), and the cells were incubated in a 5% CO₂ incubator at 37 °C. After an overnight incubation, the cells were washed with PBS followed by fixation with 4% paraformaldehyde (Sigma) for 30 min. Permeabilization was accomplished with 1% Triton X-100 in PBS for 10 min followed by blocking for 1 h with 3% bovine serum albumin (BSA) in PBS. The cells were incubated with a 1 : 200 dilution of primary antibodies against NRF2 (Santa Cruz Biotechnology) overnight at 4 °C. After washing three times with PBS, the cells were incubated with fluorescein isothiocyanate (FITC) and phycoerythrin-conjugated secondary antibodies (Santa Cruz Biotechnology) or Alexa Fluor 568 (Yellow, Abcam) in a 1 : 5000 dilution in 3% BSA-containing PBS for 1 h at room temperature in the dark. The nuclei were stained with 4,6-diamidino-2-phenyindole (DAPI, Sigma) and then examined using a Zeiss LSM700 scanning laser confocal microscope (Zen 2011; Carl Zeiss MicroImaging GHBH, Jena, Germany).

C2C12 or HSMM cells were grown on coverslips (SPL Life sciences, Phocheon-si, Korea, Cat#20018). To evaluate the protective effects of CCL2 or hCCL2 on dexamethasone-induced muscle atrophy, differentiated MTs were cotreated with recombinant CCL2 or hCCL2 (100, 200 ng/ml) and dexamethasone (200 μm) for 24 hours. C2C12 or HSMM MBs and MTs were fixed in ice-cold 4% PFA for 15 min, washed three times with PBS, and incubated in ice-cold 0.25% Triton X-100 at room temperature for 10 min [49 (link)]. Then, cells were blocked with blocking solution (1% BSA in PBS) and washed three more times. Next, cells were incubated overnight in anti-MyHC (1:400, Sigma Aldrich, St. Louis, MO, USA) at 4° C. Following three washes in PBS, the cells were incubated with Alexa 555–labeled anti-mouse IgG antibodies (1:1000, Cell Signaling, MA, USA) for 60 min. Then, cells were stained with 4, 6-diamidino-2-phenyindole (DAPI, 1:5000, Sigma Aldrich, St. Louis, MO, USA) for 10 min and washed with PBS three times. The images were produced using an LMS800 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The fusion index (%) was calculated using the following equation: 100×number of nuclei in MyHC+ MTs per total number of nuclei in MyHC+ myocytes and MTs.

For immunocytochemistry experiments, cells were grown on coverslips. After incubation, cells were fixed in paraformaldehyde 4% and permeabilized with 0.2% Triton-X100 for 10 min. After blocking with 3% BSA for 1 h, they were incubated with anti-SOX-9 ([1:100]; Millipore) overnight at 4 °C. Then, they were incubated for 1 h with the secondary antibody AlexaFluor^® 488 conjugated rabbit anti-mouse secondary antibody ([1/300]; Invitrogen; Waltham, MA, USA). Nuclei were stained with 4′,6-Diamidino-2-phenyindole (DAPI, Sigma-Aldrich; Saint Louis, MO, USA) as a control for equal cell density. The absence of a primary antibody was used as a negative control. Samples were mounted in Mowiol 40–88 (Sigma-Aldrich) and examined by a Leica TCS SP5 confocal microscope.