Cell Death Screening Using DAPI Staining: A Method for Rapid Screening of IR Induced Clonogenic Cell Death in Cancerous Cell Lines
Published: April 30, 2023
Abstract
Source: Kobayashi, D. et al. One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy. J. Vis. Exp. (2017)
This video demonstrates the methodology to screen for major cell death profiles in ionizing radiation (IR) induced cancer cell lines using DAPI staining assay. The DAPI-stained nuclei of target cells undergoing cell death exhibit distinct morphologies and can be easily identified by fluorescence microscopy.
Protocol
1. Preparation of Materials Autoclave coverslips Place coverslips in a glass beaker. Cover the glass beaker with foil. Autoclave at 121 °C at 15 psi for 20 min. Dry at 50 °C. Store at room temperature in a culture hood. Prepare the Fixation Solution: 3% paraformaldehyde + 2% sucrose in phosphate-buffered saline (PBS) To a 1,000 mL glass bottle, add 700 mL PBS and 30 g paraformaldehyde. C…
Divulgaciones
The authors have nothing to disclose.
Materials
| Cover slip | Matsunami | C013001 | |
| Paraformaldehyde | Sigma-Aldrich | P6148-1KG | |
| Sucrose | Sigma-Aldrich | 84097-250G | |
| Phosphate-buffered saline | Cosmo Bio | 16232000 | |
| Scalpel | Kai Medical | No.20, 520-A | |
| 35 mm cell culture dish | Falcon | 353001 | |
| inverted phase microscope | Shimazu | 114-909 | |
| X-ray irradiator | Faxitron Bioptics | Faxitron RX-650 | |
| square culture dish | Corning | 431110 | |
| slide glass | Matsunami | Pro-11 | |
| DAPI staining reagent | Cell Signaling | 8961S | |
| Fluorescence microscope | Nikon | ECLIPSE Ni | |
| fluorescence microscope DAPI filter | Nikon | U-FUW, BP340-390, BA420IF, DM410 | |
| fluorescence microscope lens (20×) | Nikon | Plan Apo λ 20X/0.75 | |
| fluorescence microscope lens (60×, oil) | Nikon | Plan Apo λ 60X/1.40 oil | |
| oil | Olympus | N5218600 | |
| CCD camera | Nikon | DS-Qi2 | |
| digital image acquisition software | Nikon | NIS-Elements Document | |
| lens cleaner | Olympus | OT0552 | |
| chloroform | Wako | 035-02616 |
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Cell Death Screening Using DAPI Staining: A Method for Rapid Screening of IR Induced Clonogenic Cell Death in Cancerous Cell Lines. J. Vis. Exp. (Pending Publication), e20493, doi: (2023).