Metformin-treated chondrocytes were seeded into on 15 mm cell slides (Nest Biotechnology) in 6-well plates. Cells were fixed with 4% paraformaldehyde (Beyotime, China) for 15 min and permeabilized with 0.1% Trion X-100 in PBS for 20 min. Chondrocytes were blocked with 10% BSA for 30 min and then incubated with anti-SIRT1 primary antibody (1:100, #8469, Cell Signaling Tech) overnight at 4 °C. Subsequently, cells were washed and incubated with Alexa Fluor 555-conjugated secondary antibody (1:500, A32727, Invitrogen) for 1 h at room temperature. Finally, nuclei were stained with DAPI in the dark, and fluorescence images were obtained using Fluorescence microscope (IX70, Olympus, Japan).
15 mm cell slides
Manufactured by NEST Biotechnology
Sourced in China
The 15 mm cell slides are a laboratory equipment designed for the cultivation and observation of cells. They provide a controlled environment for cell growth and microscopic analysis. The slides measure 15 millimeters in diameter and are typically made of glass or plastic materials suitable for cell culture.
Automatically generated - may contain errors
3 protocols using 15 mm cell slides
1
Immunofluorescence Assay for SIRT1
2
Immunofluorescence Staining of Chondrocytes
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HCs were seeded on 15 mm cell slides (Nest Biotechnology) in 24-well plates. 4% PFA was first used to fix cells for 15 min, and 0.5% Triton X-100 was performed to permeate cells for 20 min. After blocked by 5% BSA for 30 min, chondrocytes were incubated with a primary antibody at 4 °C overnight and then with fluorescent Alexa Fluor® 555-conjugated secondary antibody in dark at 37 °C for 1 h. The antibodies used were anti-IKKβ (1:200, Abcam Cat# ab124957, RRID: AB_10,975,710), anti-p65(1:400, Cell Signaling Technology Cat# 8242, RRID: AB_10,859,369), and fluorescent Alexa Fluor® 555-conjugated secondary goat anti-rabbit antibodies (1:500, Cell Signaling Technology Cat# 4413, RRID: AB_ 10,694,110). Fluorescence images were obtained using Nikon Ti2-E.
3
Immunofluorescence Visualization of IKK-β and p65
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Cells were plated on 15 mm cell slides (Nest Biotechnology Co. Ltd., Wuxi, China) within wells of 24-well plates, and 4% paraformaldehyde was applied to fix the cells. To permeabilize the cells, 0.5% Triton X-100 was employed. After blocking with 5% BSA for 30 min, the cells were first incubated at 4 °C with primary antibodies (against IKK-β and p65) overnight and then with a fluorescent (Alexa Fluor 555–conjugated) secondary antibody in the dark for 1 h. Fluorescence images were captured using a Nikon microscope.
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