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. © 2012 International Society for Advancement of Cytometry