Kinetic analysis of intracellular Hoechst 33342--DNA interactions by flow cytometry: misinterpretation of side population status?

We outline a simple approach involving instrument setup and calibration for the analysis of Hoechst dye 33342-loading in human cell lines for exploring heterogeneity in dye efflux efficiency and the status of side population (SP) A549 lung cancer cells. Dual excitation 488 nm/multiline UV (351-364 nm) flow cytometry was used to confirm ABCG2-specific inhibition of dye efflux using Fumitremorgin C. Transporter gene expression, assayed by qRT-PCR, confirmed higher expression of ABCG2 versus ABCB1, reiterated in a cloned subline. Coexpression of aldehyde dehydrogenase genes ranked as aldehyde dehydrogenase class 1A1 (ALDH1A1) > ALDH3A1 > ALDH3, relative expression of all genes was again reiterated in a cloned subline. Permeabilized cells were used to create red:violet (660:405 nm Em wavelengths) ratiometric references for mapping temporal changes in Hoechst 33342-DNA fluorescence in live cells. A live cell "kinetic SP gate" tracked progressive dye loading of the whole population and coapplication of the far red (>695 nm wavelength) fluorescing dye DRAQ7 enabled viable cell gating. Kinetic gating revealed a continuum for dye accumulation suggesting that SP enumeration is critically dependent upon the nonlinear relationship of the spectral shift with progressive dye-DNA binding and thus requires accurate definition. To this end, permeabilized cell reference samples permit reproducible instrument setup, guide gate boundaries for SP and compromised cells, and offer a simple means of comparing SP enumeration across laboratory sites/platforms. Our approach reports the dynamic range for the spectral shift, revealing noninformative staining conditions and explaining a source of variability for SP enumeration. We suggest that live cell kinetic sorting of all cells with the same dye:DNA load but with differences in efflux capacity can be used to explore drug resistance capability without prejudice. The SP phenotype should be regarded as a kinetic parameter and not a fixed characteristic--critical for functional assay design and the interpretation of heterogeneity.