Isolation and characterization of a Chinese hamster ovary cell mutant with improved staining for indo-1.

Abstract

Chinese hamster ovary (CHO) 10B2 cells do not stain well with indo-1 and thus cannot be used for experiments to measure intracellular calcium using this dye. We have isolated a mutant CHO cell line (CHO IS1) that stains quite well with indo-1 and that has virtually identical growth characteristics and heat sensitivity as the parent line. The mutant was isolated by sorting individual mutagenized cells with high indo-1 fluorescence and cloning them. Since it has been reported that cells with multiple drug resistance (MDR+) can pump out various fluorescent dyes, the mutant and parent lines were characterized for Hoechst 33342 staining, Adriamycin toxicity, and P-glycoprotein expression, which are markers of the MDR phenotype. P-Glycoprotein was measured with the C219 antibody using flow cytometry. Multidrug-resistant cells (CHRC5) were used as positive controls. The IS1 cells stained as well with Hoechst 33342 as fixed 10B2 cells, and much better than unfixed 10B2 cells. The IS1 cells were 10- to 30-fold more sensitive to Adriamycin than the 10B2 cells, and both cell lines were much more sensitive than the CHRC5 cells. The amount of P-glycoprotein was similar in both 10B2 and IS1 cell lines, but was about fivefold lower than the CHRC5 cells. Thus, the poor staining for indo-1 in the 10B2 cells may not be caused by the P-glycoprotein MDR pump, but by a different efflux pathway. Alternatively, the P-glycoprotein may be altered and less efficient in the CHO IS1 cells.