Axio 200

The slices without staining were obtained from the paraffin of rat esophagus, and were prepared for multiphoton microscopic imaging^{11 (link)}. The multiphoton microscopic system (Zeiss LSM 710 with upright Axio 200, Germany) was employed to image collagen and fluorescence components, separately. The channel with the wavelength range of 393–414 nm was used to presented the microstructure of collagen (green color coded), and another channel was used to exhibited the morphology of tissue components (the wavelength range of 438–708 nm, red color coded). 12-bit pixel depth and 2.56 μs per pixel were set. The fast fourier transform analysis was employed to reveal the collagen content of the esophageal stricture model.

mBMSCs were plated in a 12-well plate. Once the culture reached about 50 % confluence, the medium was replaced with 0.5 ml of new medium containing 1:10 diluted lentiviral particles and 2 μg/ml Polybrene (Hexadimethrine bromide), and incubated for 8 h. Afterwards the cell surface was washed with PBS three times and 1 ml of normal medium was added in each well. After 48 h, mBMSCs expressing green fluorescent protein (GFP) were sorted out using a fluorescence activated cell sorting (FACS) machine (BD FACSAria II cell sorter). Expression of GFP was confirmed under a fluorescent microscope (Zeiss Axio 200) and secretion of hVEGFa, hSDF1a, and hTNFa was determined by enzyme-linked immunosorbent assay (ELISA) kits (R&D systems).

Intracellular ROS were detected with 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA; Molecular Probes, OR, USA), according to the manufacturer’s instructions. Myotubes were differentiated in 24-well cell culture plates (SPL, Seoul, Korea) with L6 myoblasts (1 × 10⁴/well) for microscopic image analyses or in 96-well cell culture plates (SPL, Seoul, Korea) with L6 myoblasts (5 × 10³/well) for fluorometric analyses. Differentiated myotubes were cultivated in serum-free DMEM with or without test agents for 18 h and stained with 5 μM H2DCFDA for 30 min in a humidified incubator at 37 °C with 95% air and 5% CO₂. Image analyses were performed with stained cell images taken by an inverted fluorescence microscope (Axio200, Carl Zeiss, Oberkochen, Germany). Fluorometric analyses were done using an Absorbance Microplate Reader (SpectraMAX^®, Molecular Devices, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 519 nm.