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A sensitive assay for 6-thioguanine-resistant lymphocytes
A Norman1, J C Mitchell, K S Iwamoto
1Department of Radiological Sciences, University of California, Los Angeles 90024.
Mutation Research
|May 1, 1988
Summary
This study defines 6-thioguanine (TG) resistant variants (V) using a novel 72-hour assay. This method significantly increases the detection of TG resistant variants and gamma-ray induced variants in lymphocytes.
Area of Science:
- Cell Biology
- Genetics
- Radiation Biology
Background:
- 6-thioguanine (TG) resistance is a key marker in cellular studies.
- Conventional assays for TG resistance have limitations in detecting slow-growing variants.
Purpose of the Study:
- To develop and validate a prolonged incubation method for identifying 6-thioguanine (TG) resistant variants (V).
- To enhance the sensitivity of detecting radiation-induced TG resistance in lymphocytes.
Main Methods:
- Cells were incubated for 72 hours in medium containing 0.2 mM TG.
- Cytokinesis was blocked with cytochalasin B (CB) after 30 hours.
- Nuclear division was assessed by counting cells with 2 or more nuclei.
Main Results:
- The 72-hour incubation period identified an order of magnitude more TG resistant variants (V) compared to conventional 30-40 hour assays.
- In the 0-0.5 Gy gamma-ray dose range, 80 V per 1000 surviving lymphocytes per Gy were detected, a two-order of magnitude increase over previous reports.
Conclusions:
- The extended incubation assay significantly improves the detection of slow-growing TG resistant variants.
- This enhanced assay provides a more sensitive method for quantifying DNA damage response to ionizing radiation.