86 A Garozzo et aL Antiviral Research 89(2011)83-88 Fig.3.Acridine orange staining of MDCK cells:untreated(DMSO)(A).treated with 100 and 10nM bafilomycinA1 for 1h(B and C):with .01TTO for 4h(D)and for 1h(E): with 0.01%terpinen-4-ol for 4h(F):treated with 0.01 TTO for 4 h followed by washing and incubation without TTO (G). controls.For control experiments,only DMSO was added to the cence was indicative of a diminished concentration of ao in the medium instead of the compounds. cells. The compounds terpinen-4-ol,terpinolene,and o-terpineol were used at the concentration of 0.01%(v/v),0.005%(v/v),and 2.10.Reversibility of drug effect 0.02%(v/v),respectively. The fluorescence signal depends on the AO accumulation in To study the possible reversibility of the TTO effect,MDCK cells acidified cellular compartments,therefore an increased fluores- were incubated at 37C for 4h with TTO,and terpinen-4-ol (0.01,86 A. Garozzo et al. / Antiviral Research 89 (2011) 83–88 Fig. 3. Acridine orange staining of MDCK cells: untreated (DMSO) (A), treated with 100 and 10 nM bafilomycin A1 for 1 h (B and C); with 0.01% TTO for 4 h (D) and for 1 h (E); with 0.01% terpinen-4-ol for 4 h (F); treated with 0.01% TTO for 4 h followed by washing and incubation without TTO (G). controls. For control experiments, only DMSO was added to the medium instead of the compounds. The compounds terpinen-4-ol, terpinolene, and -terpineol were used at the concentration of 0.01% (v/v), 0.005% (v/v), and 0.02% (v/v), respectively. The fluorescence signal depends on the AO accumulation in acidified cellular compartments, therefore an increased fluores￾cence was indicative of a diminished concentration of AO in the cells. 2.10. Reversibility of drug effect To study the possible reversibility of the TTO effect, MDCK cells were incubated at 37 ◦C for 4 h with TTO, and terpinen-4-ol (0.01,