The viability of cells seeded onto the hydrogels was evaluated by Live/Dead Cell Staining Kit purchased from Tianjin Wei Kai Biological Engineering Co., Ltd. (Tianjin, China), and performed according to the manufacturer's instructions. Briefly, 1 mm thick discs of hydrogels were cut to be fit in the 24-well cell culture plate. The discs were soaked in 75% ethanol overnight for sterilization and then washed by PBS for three times. The sterilized samples were immersed in the culture medium for 24 h. The medium was subsequently removed, and the cells prepared above were seeded onto the hydrogels and incubated at 37 °C with a concentration of 5 × 104 cells per sample. The seeded cell and hydrogel materials were treated with or without PEMFs. After a 7 day incubation, the samples were incubated in 400 μL culture medium with 2 μM calcein-AM (staining live cells) and 4 μM ethidium homodimer-1 (staining for dead cells) for 45 min, then examined under a Leica DM16000B microscope. Images were captured using a Leica DFC550 digital camera and analyzed using a Leica LAS V4.6 software.
Dm16000b microscope
Manufactured by Leica
The DM16000B is a high-performance microscope from Leica. It is designed for advanced imaging and analysis in laboratory settings. The DM16000B features a modular optical system, enabling versatile configurations for a wide range of applications. Its core function is to provide precise, high-resolution imaging capabilities for researchers and scientists.
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2 protocols using dm16000b microscope
1
Viability Evaluation of Cell-Seeded Hydrogels
2
4D Live Cell Imaging of Fluorescently Labeled Cells
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Cells were plated onto 4 well Lab-Tek II covered chamber slides at 50 % confluency. Prior to imaging, media was replaced with phenol-red free DMEM with 25 mM HEPES (Invitrogen) and 10 % FBS. Slides were incubated in a Live Cell Imaging humidified chamber that was maintained at 37 °C and 5 % CO2. LCM was performed using a Leica DM16000B microscope described above. Volocity Imaging software (v4.3.2; PerkinElmer) was used as acquisition software. 3D reconstruction analyses were performed by Imaris software v. 7.6 (Bitplane, Inc.). The excitation for GFP was 491 nm and the filter was 525/50 nm. The excitation for mCherry was 561 nm and the filter was 595/50 nm.
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