Chambered cell culture cover glasses

Cells were seeded on 1.8 cm 2 chambered cell culture cover glasses (Nunc, UK) at a density of 3 x 10 4 cells/well. Cells were grown for 24-48 hours and then incubated for 2 hours with 100 µg/ml transferrin-AF488, 2 µM lacCer-BODIPY or 1 mg/ml fluorescein isothiocyanatedextran (150 kDa) in the presence of 250 µg/ml PP-50/AF647. Subsequently, cells were incubated for 15 minutes with 5 µg/ml Hoechst 33342; this permeable dye is used to evaluate nuclear morphology as it emits fluorescence when bound to DNA. Cells were washed twice with PBS and extracellular labelled molecules were quenched using 0.4 % trypan blue [20] (link). Cells were washed again with PBS and left in growth media. Cells were analysed using a TCS SP5 inverted laser scanning microscope (Leica, Germany). An argon laser was used to visualise transferrin-AF488, lacCer-BODIPY, and fluorescein isothiocyanate-dextran (emission at 488 nm and an emission filter set at 505-555 nm). To visualise PP-50/AF647, a helium neon laser was used (emission at 633 nm and emission filter set at 650-700 nm). H33342 stained nuclei were excited using a diode laser emitting at 405 nm. Images were taken sequentially.

Cells were seeded on 1.8 cm 2 chambered cell culture cover glasses (Nunc, UK) at a density of 3 x 10 4 cells/well and grown for 24-48 hours. Cells were then transduced with CellLight reagents according to the manufacturer's instructions. Briefly, cells were incubated for 16 hours with 30 µl of CellLight solution with baculovirus containing either early endosomes Rab5a-GFP, late endosomes Rab7a-GFP or lysosomes Lamp-1-GFP. After this period, cells were washed twice with PBS, incubated with 250 µg/ml PP-50/AF647 for 2 and 24 hours and finally incubated for 15 minutes with 5 µg/ml H33342. Cells were washed twice with PBS and extracellular labelled molecules were quenched using 0.4 % trypan blue. Cells were washed again with PBS and left in growth media. Cells were analysed using a TCS SP5 inverted laser scanning microscope (Leica, Germany). An argon laser was used to visualise the GFP labelled early endosomes, late endosomes and lysosomes (excitation at 488 nm and emission filter set at 505-555 nm). To visualise PP-50/AF647, a helium neon laser was

Cells were seeded on 1.8 cm 2 chambered cell culture cover glasses (Nunc, UK) at a density of 3 x 10 4 cells/well and grown for 24-48 hours. Cells were then incubated with 50 mM FITCtrehalose in the presence or absence of 250 µg/ml PP-50 for 2, 4, and 6 hours. FITCtrehalose and PP-50 solutions were prepared in DMEM. Cells were washed twice with PBS and extracellular labelled molecules were quenched using 0.4 % trypan blue [30] (link). Cells were washed again with PBS and left in growth media. Cells were incubated for 15 minutes with 5 µg/ml PI and 5 µg/ml H33342 then incubated in growth media and analysed using a TCS SP5 inverted laser scanning microscope (Leica, Germany). An argon laser was used to visualise FITC-trehalose (emission at 488 nm and an emission filter set at 505-555 nm). To visualise propidium iodide, a helium neon laser was used (emission at 633 nm and emission filter set at 650-700 nm). H33342 stained nuclei were excited using a diode laser emitting at 405 nm. Images were taken sequentially.