Mode of dye loading affects staining outcomes of fluorescent dyes in astrocytes exposed to multiwalled carbon nanotubes

The effects of multiwalled carbon nanotubes (MWCNTs) and fullerene C60 (C60) on staining of rat astrocytes by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1), 3,3′-dihexyloxacarbocyanine, iodide (DiOC6(3)), rhodamine 123 (Rho 123) and fluorescein diacetate (FDA) were investigated. Probe indications were characterized and probe staining performed both in an “in situ” and “ex situ” manner on MWCNTs- or C60-exposed cells were analyzed by flow cytometry. JC-1 and DiOC6(3) staining were found good indicators of mitochondrial membrane potential (ΔΨm). While ΔΨm contributed to Rho 123 uptake, intracellular accumulation of Rho 123 was largely determined by the function of P-glycoprotein, a cell membrane bound efflux protein. Multidrug-resistance associated protein, another efflux protein, was found to determine cellular FDA staining. Decreased JC-1 and DiOC6(3) uptake were observed in MWCNTs-exposed cells but could not be attributed to ΔΨm disruption. In contrast, cellular staining of Rho 123 and FDA was enhanced after MWCNT exposure. Mode of dye loading was found to significantly affect the outcome of cellular dye staining after MWCNT exposure. Compared with MWCNTs, C60 generally exerted insignificant influence on the staining of all probes. The possible reasons for these findings and their implications are discussed.