Toxicology 160 (2001) 5–10 Gel microdrop flow cytometry assay for low-dose studies of chemical and radiation cytotoxicity K.T. Bogen a, *, L. Enns b , L.C. Hall a , G.A. Keating a , M. Weinfeld b , G. Murphy a , R.W. Wu a , F.N. Panteleakos a a Health and Ecological Assessment Diision (L.396), Lawrence Liermore National Laboratory, 7000 East Aenue, Liermore, CA 94550 9900, USA b Cross Cancer Institute, Edmonton, Alta, Canada Abstract Low-level cytotoxicity may affect low-dose dose – response relations for cancer and other endpoints. Conventional colony-forming assays are rarely sensitive enough to examine small changes in cell survival and growth. Automated image-analysis techniques are limited to ca. 10 4 cells/plate. An alternative method involves encapsulation of single proliferating cells into ca. 35 – 75-m-diameter agarose gel microdrops (GMDs) that are randomly grouped, differen- tial exposure of these groups, culture at 37°C for 3–5 days, and finally GMD analysis by flow cytometry (FC) to determine the ratio of GMDs containing multiple versus single cells as a measure of clonogenic survival. This GMD/FC assay was used to examine low-dose cell killing induced by a cooked-meat mutagen/rodent-carcinogen (MeIQx) in DNA-repair-deficient/metabolically-sensitive CHO cells. Results of conventional colony-forming assays using up to 30 replicate plates indicate a shouldered, threshold-like dose – response; in contrast, those obtained using the GMD/FC assay suggest ‘hypersensitivity’-like nonlinearity in dose – response. The GMD/FC assay was also applied to human A549 lung cells after GMD-encapsulation and  radiation followed by culture for a total of 4 days, to examine survival after exposure to 100 cGy delivered at a relatively low dose rate (0.18 cGy/min). Dose–re- sponse for clonogenic growth was again observed to be reduced with apparent nonlinear suggesting hypersensitivity between 0 and 50 cGy, insofar as doses of 5 and 10 cGy appear to be ca. fivefold more effective per unit dose than the 50- or 100-cGy doses used. The GMD/FC assay may thus reveal low-dose dose – response relations for chemical and radiation effects on cell proliferation/killing with implications for low-dose risk assessment. © 2001 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Assay; Cell killing; Chemical mutagen; Flow; Low-dose; Low-LET radiation; Microdrop; Survival www.elsevier.com/locate/toxicol Abbreiations: CHO, Chinese hamster ovary; GMD, gel microdrop; FC, flow cytometry; MeIQx, 2-amino-3,8-dimethylimi- dazo[4,5-f ]quinoxaline (CAS c 77500-04-0); RT, room temperature (ca. 25°C); S.E., standard error. * Corresponding author. Tel.: +1-925-4220902; fax: +1-925-4243255. E-mail address: bogen@llnl.gov (K.T. Bogen). 0300-483X/01/$ - see front matter © 2001 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S0300-483X(00)00432-7