The gels were placed in a 48-well plate and soaked in low-glucose DMEM for 24 h. The medium was removed, and 1 ml of BMSC medium containing 1 × 105 cells was seeded onto each gel before incubation. The culture medium was replaced every 2 days. After 5 days of culture, the gels were stained using the live–dead staining kit (Solarbio), according to the manufacturer’s instructions. The stained samples were observed under a laser scanning confocal microscope (Nikon, Japan).
Live dead staining kit
Manufactured by Solarbio
Sourced in China
The Live/Dead Staining Kit is a tool used to differentiate between live and dead cells in a sample. It utilizes fluorescent dyes that bind to specific cellular components, allowing for the visualization and quantification of viable and non-viable cells.
Automatically generated - may contain errors
Lab products found in correlation
Laser scanning confocal microscope, by Nikon (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Inverted fluorescence microscopy, by Olympus (1 mentions)
Hematoxylin eosin staining kit, by Solarbio (1 mentions)
Triethylamine, by Solarbio (1 mentions)
Heparin sodium, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Cck 8, by Solarbio (1 mentions)
5 protocols using live dead staining kit
1
BMSC Viability Evaluation in 3D Gels
2
Evaluation of Zirconia Cytotoxicity on MC3T3-E1 Cells
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MC3T3-E1 cells were seeded at a density of 1× 104 cells/well in a 24-well plate. At each culture period (1 and 3 d), a live/dead staining kit (Solarbio, China) was utilized to assess the cytotoxicity of zirconia specimens. Calcein-AM was a staining reagent for fluorescently labelling living cells with green fluorescence, and its working concentration was 1 μΜ. In addition, PI (3 μΜ) only stained dead cells and excited red fluorescence. Finally, dyed cells were visualized through fluorescence microscope (Olympus, Japan).
3
Cytotoxicity Evaluation of ASA, nHAP, and ASA-nHAP
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The cytotoxicity was assessed with a Live/dead Staining Kit (Solarbio, Beijing, China). The MC3T3-E1 cells were seeded in a 96-well plate at a density of 3,000 per well (n = 3). After 24 h, the original CM was replaced by 100 μL fresh CM, and the extractions of ASA, nHAP, and ASA-nHAP group for further incubation. The original CM and extractions were replaced with 100 μL of live/dead cell staining reagent after 1, 3, and 5 days and stained for 30 min. The stained cells were observed and imaged by a X171 Inverted Fluorescence Microscope (Olympus, Japan), and then analyzed cell viability.
4
Cell Viability Evaluation of hGMSCs/Matrigel Mixture
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The cell viability of the hGMSCs/Matrigel mixture was assessed with a live/dead staining kit (Solarbio, Beijing, China). The experiments were performed according to the kit instructions. After seeding, the mixture was stained on days 1, 3, 5, and 8, followed by submersion in staining solution for 40 min in the dark. Cell viability was visualized via inverted fluorescence microscopy (Olympus, Tokyo, Japan). Viable cells were marked with Calcein-AM (Ex/Em = 490/515 nm), whereas dead cells were marked with propidium iodide (PI) (Ex/Em = 535/617 nm). The cells were counted under a microscope at 200× magnification (n = 6 per group). Three fields of view were randomly selected for each well of the 24-well plate, and the data were averaged. The dead cell ratio (DCR) was calculated as follows: DCR = the number of dead cells/the total number of cells×100%.
5
Chitosan-based Biomaterial Characterization
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CS (molecular weight: 100 kDa, deacetylation degree: 85%) was provided by Jinan Haidebei Biotechnology Co., Ltd., China. CCK-8, live/dead staining kit, lactic dehydrogenase kit, hematoxylin-eosin (H&E) staining kit, and other chemical reagents (e.g., N,N-dimethylformamide, triethylamine, ethanol, acetic acid, glutaraldehyde) were obtained from Beijing Solarbio Science & Technology Co., Ltd., China.1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylenegl-ol)](DSPE-PEG-NHS) was purchased from Guangzhou Carbon Water Biotechnology Co., Ltd., China. Thrombin, heparin sodium, and lysozyme were provided by Sigma-Aldrich. Male Sprague-Dawley rats (weight: 250–300 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd., China.
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