BHK cells co-expressing GFP-tagged Ras anchors and the empty vector pC1 or RFP-tagged Ras anchors were grown to ∼85% confluency and washed with 2× Hepes buffer. The cells were then incubated in Hepes buffer containing 2 mM (N-ethyl maleimide [NEM]) for ∼90 min to induce blebbing (83 (link)). GPMVs containing GFP- and RFP-tagged Ras anchors (or GFP-tagged Ras anchors with pC1) were then incubated in Hepes buffers containing various percentages of deionized water (hypotonic conditions) or concentrations of NaCl (hypertonic conditions) for 5 min.
GFP fluorescence was visualized using a Nikon TiE wide-field microscope using a 60× oil-emersion PLAN-Apo/1.4 numerical aperture lens (25 (link),26 (link)). The fluorescence lifetime of GFP was measured using a Lambert FLIM unit attached to the wide-field microscope. GFP was excited using a sinusoidally stimulated and modulating 3-W 497-nm light-emitting diode at 40 Hz. At least 20 vesicles were imaged and the fluorescence lifetime values were pooled and averaged. Statistical significance was evaluated using one-way ANOVA, with * indicating P < 0.05.
GFP fluorescence was visualized using a Nikon TiE wide-field microscope using a 60× oil-emersion PLAN-Apo/1.4 numerical aperture lens (25 (link),26 (link)). The fluorescence lifetime of GFP was measured using a Lambert FLIM unit attached to the wide-field microscope. GFP was excited using a sinusoidally stimulated and modulating 3-W 497-nm light-emitting diode at 40 Hz. At least 20 vesicles were imaged and the fluorescence lifetime values were pooled and averaged. Statistical significance was evaluated using one-way ANOVA, with * indicating P < 0.05.
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