Apoptosis Assays

Question 1

Sucrose Velocity Sedimentation

Answer 1

Dose Range
Ssb > 5 Gy
Dsb > 15 Gy

Technique
Larger DNA fragments sediment to a greater extent

Limitations
Insensitive to clinically-relevant low radiation doses

Question 2

Filter Elution

Answer 2

Dose Range
Ssb > 1 Gy (alkaline elution)
Dsb > 5 Gy (Neutral elution)

Technique
Smaller DNA fragments elute more quickly through a filter of defined pore size

Limitations
Uncertain which DNA lesions are being detected

Question 3

Nucleoid sedimentation

Answer 3

Dose Range
Ssb 1-20 Gy

Technique
Irradiatied cells show altered DNA supercoiling within nucleus

Limitations
Uncertain which DNA lesions are being detected

Question 4

Pulse-field gel electrophoresis (PFGE)

Answer 4

Dose Range
Dsb > 5-10 Gy

Technique
Allows for resolution of DNA-dsb, which can be quantified by relative migration within the gel

Limitations
Uncertain effects of DNA conformation. High number of cells in S phase may bias results of assay

Question 5

Comet Assay

Answer 5

Dose Range
Ssb >1 Gy (alkaline lysis)
Dsb > 2 Gy (neutral lysis)

Technique
Following lysis, individual nuclei are subjected to agarose gel electrophoresis. The DNA that moves out of the nucleus (head) to form the “gail” of the comet is quantified to provide a measure of DNA damage

Limitations
Requires image analysis system to quantify DNA damage. Increased numbers of cells in S phase may bias assay

Question 6

Fluorescence in situ hybridization (FISH)

Answer 6

Dose Range
Doses > 1 Gy

Technique
Chromosome-specific probes, which can be detected with a fluorescent ligand, are used to identify radiation-incuced translocations

Limitations
May be difficult to interpret in tumor cells that contain translocations prior to irradiation

Question 7

Premature chromosome condensation (PCC)

Answer 7

Dose Range
Doses > 1 Gy

Technique
An irradiated interpose cell is fused to a mitotic cell. The chromosomes in the interphase cell undergo premature condensation, allowing radiation-induced chromosome damage to be scored

Limitations
May be difficult to interpret in tumor cells that contain chromosome aberrations prior to irradiation

Question 8

gamma-H2AX intranuclear foci

Answer 8

Dose Range
Doses > 0.05 Gy

Technique
Immunofluorescence microscopy or flow cytometry using an antibody to gamma-H2AX phosphoprotein

Limitations
Requires image analysis system. No standard for size of type of foci to count as DNA breaks