Dmi8 microscope

Images were obtained using a Leica DMi8 microscope with a PE4000 LED light source, DFC9000GT camera, and LAS X imaging software. A549 cells were seeded in 3 cm tissue culture plates at a density of 50,000 cells per well in DMEM containing fluorescent organic salts at indicated concentrations. The cells were incubated for 2 days at 37 °C with 5% CO₂ until the day of imaging. For live cell imaging, the media was aspirated, and the cells were washed with phosphate buffered saline (PBS, Sigma-Aldrich) 5 times before being imaged in PBS.
For colocalization analysis, A549 cells were grown on 0.5 mm coverslips placed in 3 cm tissue culture plates containing media for 3 days. Cells were then fixed by aspirating media, washing with PBS 5 times, then submerging the coverslip in cold methanol and incubating on ice for 15 minutes. The fixed cells were stained with 1 µM 2′-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]−2,5′-bi-1H-benzimidazole trihydrochloride trihydrate (Hoechst 33342, Invitrogen) for 5 minutes, washed with PBS, and then incubated with 15 µM of 3,6-diamino-9-(2-(methoxycarbonyl)phenyl chloride (Rhodamine123) and 1 µM CyPF₆ for 15 minutes before being washed and mounted to slides with Fluoromount-G (Invitrogen). Cells were analyzed using a Leica DMi8 microscope with a PE4000 LED light source, DFC9000GT camera, and LAS X imaging software.

Two-dimensional cultures were imaged using a Leica DMI8 microscope using 10× objective. Chanel filter Da/FI/TX was used which uses EX:394-412;483-501;562-588, DC:535;505;595 and EM: 441-471;512-548;600-660 for mCherry, mVenus and mCerulean.
For quantifying cell survival after picoliter deposition, cells were imaged for four days at 30 min intervals, with a Leica DMI8 microscope in phase-contrast mode at 10× magnification, 37 °C and 5.0% CO₂. Imaging was initiated four hours after the generation of colonies. 3D organoids were imaged with the Leica SP8X confocal microscope using a 20× objective. 515 nm and 587 nm wavelengths for excitation, 527 and 610 wavelengths for emission of mVenus and mCherry were used, respectively.

Sheep primary myoblasts were cultured to 100% fusion and induced to form myotubes, and paraformaldehyde (4%) was used to fix the cells on ice for 10 min. Using 0.25% Triton X-100 permeabilized myoblasts for 10 min and blocked with 1% bovine serum albumin for 1.5 h. Primary antibody (MHC) was used to incubate myoblasts overnight at 4 °C, and then corresponding secondary antibody was used to incubate myoblasts for 1 h at room temperature. Finally, DNA was stained with DAPI for 3 min and images were acquired under a DMi8 microscope (Leica, Germany). Finally, DNA was stained with DAPI for 3 min and images were acquired under a DMi8 microscope (Leica, Germany).