Uplanapo ir 60x 1.20w water immersion objective lens

A431 cells were grown on an eight well microscope slides (BD Biosciences, NJ, USA). To determine if LS662 preferentially stains dead cells, three conditions of A431 cells were incubated with 1 μM LS662 or 1 μM cypate for 2 h: healthy cells (cell in medium with LS662), dying cells (cell incubated with PBS with LS662), and dead cells (cell pretreated with 50% ethanol in PBS for 30 min, with LS662 added after the treatment with ethanol). After treatment, the cells were rinsed with PBS, mounted with Vectashield (Vector Laboratories, CA, USA), and coversliped. The slides were imaged using a FV1000 confocal microscope with an UPLanApo/IR 60X/1.20W water immersion objective lens (Olympus, PA, USA) at 633 nm (LS662) or 785 nm (cypate) excitation, and fluorescence was detected at 690/50 nm (LS662), or 850 LP (cypate).

A431 cells were grown in 96 well plates (BD Biosciences, NJ, USA). The cells were treated with 1 μM of LS662 for 1–8 h, in either acidic (pH 6.4) or neutral (pH 7.4) medium. Acidic medium was made by adding 25 μL of 1 M HCl per 1 mL of medium. For blocking studies, 10 mM sodium azide, an active transport inhibitor, was co-incubated with the cells. After treatment, the cells were rinsed with PBS and imaged using a BX-51 epifluorescent microscope (Olympus, PA, USA). A cy5 U-N41008 (Chroma Technology Corp, VT, USA) filter cube was used for excitation at 620/60 nm and emission collection at 700/75 nm.
4T1/luc cells were grown in an 8 well slide (BD Biosciences, NJ, USA). The cells were treated with 10 μM of LS662 or 1 μM of cypate for 2 or 8 h, in either acidic (pH 6.4) or neutral (pH 7.4) medium. After treatment, the cells were rinsed with acidic or neutral medium and imaged using a FV1000 confocal microscope with an UPLanApo/IR 60X/1.20W water immersion objective lens (Olympus, PA, USA) at 633 nm (LS662) or 785 nm (cypate) excitation laser, and fluorescence was detected at 690/50 nm (LS662), or 850 LP (cypate).