Cell Death Screening Using DAPI Staining: A Method for Rapid Screening of IR Induced Clonogenic Cell Death in Cancerous Cell Lines

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

1. Autoclave coverslips
   1. Place coverslips in a glass beaker.
   2. Cover the glass beaker with foil.
   3. Autoclave at 121 °C at 15 psi for 20 min.
   4. Dry at 50 °C. Store at room temperature in a culture hood.
2. Prepare the Fixation Solution: 3% paraformaldehyde + 2% sucrose in phosphate-buffered saline (PBS)
   1. To a 1,000 mL glass bottle, add 700 mL PBS and 30 g paraformaldehyde.
      CAUTION: Paraformaldehyde is corrosive, wear appropriate gloves.
   2. Dissolve paraformaldehyde completely by heating to 80 °C usin....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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