Flow cytometry techniques for studying cellular thiols
Cellular thiols, and especially glutathione, act as scavenger nucleophiles and can protect against toxicity, mutagenicity, or transformation by ionizing radiation and many carcinogens. Development of a rapid assay to quantitate the cellular content of thiols could thus be useful in assessing or predicting cellular risk to damage. Several fluorescent thiol-reactive drugs, usually maleimide or bromobimane derivatives, have been described for use in histopathology. Most of these agents do not distinguish between protein and nonprotein thiols, and virtually all of these fluorescent stains have normally been used after fixation of the cells or tissues. We have found that some of the probes will, however, rapidly penetrate and bind within viable cells with little associated cytotoxicity; the amount bound can be easily quantified using flow cytometry. We have used several of these agents, in conjunction with fluorescence-activated cell sorting in V79 spheroids, to examine the thiol content of cells as a function of their depth or position in the spheroid. Additionally, the radiation response of cells from different depths as been assessed following addition of exogenous thiols including glutathione and WR-2721, or after treatment with thiol-depleting agents, including DL-buthionine-S,R-sulfoximine (BSO), diethylmaleate (DEM), and dimethylfumarate (DMF). Our studies indicate that examination of the thiol content and radiation response of the sorted cells provides an improved understanding of the modes of action of these compounds.
- Research Organization:
- Johns Hopkins Oncology Center, Baltimore, MD
- OSTI ID:
- 5653342
- Journal Information:
- Radiat. Res.; (United States), Journal Name: Radiat. Res.; (United States) Vol. 95:3; ISSN RAREA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
560121* -- Radiation Effects on Cells-- External Source-- (-1987)
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMAL CELLS
ANIMALS
AROMATICS
BIOCHEMISTRY
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CARBOXYLIC ACIDS
CELL FLOW SYSTEMS
CHEMISTRY
CONNECTIVE TISSUE CELLS
DRUGS
DYES
FIBROBLASTS
FLUORESCEIN
GLUTATHIONE
HAMSTERS
HYDROXY ACIDS
HYDROXY COMPOUNDS
MAMMALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDES
PHENOLS
POLYPEPTIDES
POLYPHENOLS
PROTEINS
RADIATION EFFECTS
RADIOPROTECTIVE SUBSTANCES
RADIOSENSITIVITY EFFECTS
RODENTS
SOMATIC CELLS
SPHEROIDS
SULFOXIDES
THIOLS
VERTEBRATES