Home › Cell Biology › One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy
Cell Biology JoVE (Open Access) Citable · DOI

One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

DOI: 10.3791/56338-v
What you'll learn
  • ✓ Classify radiation-induced cell death modes by nuclear morphology using DAPI staining
  • ✓ Perform one-step fluorescence microscopy assay on irradiated cell cultures
  • ✓ Distinguish apoptosis, mitotic catastrophe, and senescence by nuclear phenotypes
Protocol

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effects of IR on malignant tumors and normal tissues. Here, we describe a one-step assay for assessing the major modes of IR-induced clonogenic cell death based on morphology of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei visualized by fluorescence microscopy.

Difficulty
intermediate
Total time
~3-4 days (including culture growth, irradiation, fixation, imaging, and analysis)
Model organism
Cultured mammalian cells (specific line not specified in abstract)
Biosafety
BSL-1

Steps

1
Culture cells and deliver ionizing radiation

Grow adherent cells in culture and expose to controlled ionizing radiation dose. Allow cells to recover for specified period post-irradiation.

▶ 01:31
2
Fix cells and apply DAPI nuclear stain

Fix irradiated cells using standard fixation protocol and stain nuclei with DAPI dye to visualize chromatin morphology.

▶ 02:53
3
Classify cell death modes by nuclear morphology

Image stained cultures by fluorescence microscopy and categorize nuclei according to characteristic morphologies associated with apoptosis, mitotic catastrophe, senescence, or surviving clones.

▶ 04:44
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