The SA-β-gal activities of young (passage number 3) and old (passage number 7) HASMCs were detected using a SA-β-gal staining kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol. The number of blue cells (SA-β-gal-positive cells) and the total number of cells in six randomly chosen fields were counted to calculate the percentage of senescent cells using an ordinary optics microscope (Olympus Corporation, Tokyo, Japan). Percentage of senescent cells = (number of SA-β-gal positive cells/number of total cells) x 100%.
Ordinary optics microscope
Manufactured by Olympus
Sourced in Japan
The Ordinary optics microscope is a laboratory instrument designed for magnifying and observing small objects. It utilizes a combination of lenses to provide a clear and detailed view of specimens. The core function of this microscope is to enable visual inspection and examination of materials at a microscopic level.
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6 protocols using ordinary optics microscope
1
Senescence-Associated β-Galactosidase Assay
2
Cell Adhesion Assay on Fibronectin
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A 24-well plate was coated with fibronectin (BD Biosciences, California, USA) at 37 °C for 1 h, followed by blocking with 1% FBS for 30 min. Then, 3 × 104 cells were seeded in each well and incubated at 37 °C for 1 h, rinsed in phosphate-buffered saline (PBS) buffer, fixed with formalin, stained with hematoxylin, and enumerated by an ordinary optics microscope (Olympus).
3
Cell Invasion Assay Protocol
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Cells (0.1–1 × 106) were resuspended in 200 μL serum-free medium and seeded in the 24-well transwell upper chambers (Corning, Corning, NY, USA). A total of 500 μL RPMI 1640 medium containing 10% FBS was added into the lower chambers. After culturing for 12–48 h, cells on the membrane were fixed with formalin, stained with H&E, and then calculated and photographed using the ordinary optics microscope (Olympus). Invasion assays were performed according to the same procedures with the diluted Matrigel (Corning) (RPMI 1640: Matrigel = 5:1) spread on the inside bottom of the transwell upper chambers.
4
Transwell Migration and Invasion Assays
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The transwell migration and invasion assays were performed as described previously [23 (link)]. The images were acquired by using an ordinary optics microscope (Olympus).
5
Wound Healing Assay Protocol
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The cells were seeded in a six-well plate and cultured to 90% confluence. The wounds were scratched with a sterile 10 μL pipette tip on a cell monolayer. The cells were cultured in a serum-free medium for 48 h. The images were acquired using an ordinary optics microscope (Olympus, Japan) at 0 h and 48 h. The wound-healing percentage was calculated according to this formula: (the size of the wound at 0 h − the size of the wound at 48 h)/the size of the wound at 0 h.
6
Transwell Invasion Assay Protocol
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Cells (0.1-1×10 6 ) were resuspended in 200µL serum-free medium and seeded in the 24-well transwell upper chambers (Corning, NY, USA). 500µL RPMI-1640 medium containing 10% FBS was added into the lower chambers. After culturing for 12-48h, cells on the membrane were xed with formalin, stained with hematoxylin and then calculated and photographed using the ordinary optics microscope (Olympus). Invasion assays were performed according to the same procedures with the diluted Matrigel (Corning) (RPMI-1640: Matrigel = 5:1) spread on the inside bottom of the transwell upper chambers.
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