A novel flow cytometric technique for drug cytotoxicity gives results comparable to colony-forming assays.
BACKGROUND
Drug sensitivity is commonly determined by assays that utilize colony formation to discriminate between surviving and lethally treated cells. These assays require cells with high plating efficiency that form discernible colonies, are time-consuming and laborious, and require manual counting of large numbers of colonies. To overcome these drawbacks, we developed a flow cytometric technique that assays survival of proliferative capacity in cultured cells.


METHODS
Labeling with bromodeoxyuridine for 72 h followed by bivariate Hoechst 33258/ethidium bromide flow cytometry allows discrimination of nonproliferating cells from those that have undergone one to three divisions. Addition of an internal standard, chicken erythrocyte nuclei, permits determination of total cell number. To validate our assay, we used flow and colony-forming assays to determine the sensitivity of cell lines derived from Werner syndrome patients and unaffected individuals to 4-nitroquinoline-1-oxide (4NQO) and camptothecin.


RESULTS
The flow and colony-forming assays yielded comparable sensitivity for each drug and essentially identical increases in drug sensitivity exhibited by Werner syndrome cells.


CONCLUSIONS
Our results indicate that the flow assay is a less laborious surrogate for colony-forming assays. The flow technique will also facilitate the analysis of drug sensitivity in cells that are not amenable to colony-forming assays.
Department of Pathology, University of Washington, PO Box 357705, Seattle, WA 98195-7705, USA. MartinP@medicine.washington.edu
Martin
M
Poot
John R
JR
Silber
Peter S
PS
Rabinovitch
D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology; D000963:Antimetabolites; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001562:Benzimidazoles; D001973:Bromodeoxyuridine; D002166:Camptothecin / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D008214:Lymphocytes / Q000166:cytology; D013234:Stem Cells / Q000473:pathology*; D014898:Werner Syndrome / Q000473:pathology*
D000963:Antimetabolites; D000972:Antineoplastic Agents, Phytogenic; D001562:Benzimidazoles; D002273:Carcinogens; D005456:Fluorescent Dyes; D015112:4-Nitroquinoline-1-oxide; D001973:Bromodeoxyuridine; C017807:bisbenzimide ethoxide trihydrochloride; D002166:Camptothecin