Lipid raft staining and immunofluorescence imaging

To label lipid rafts, two lipid raft-specific dyes were used, a Vybrant Alexa Fluor 555 lipid raft labeling kit (Invitrogen) and BODIPY FL C₅-ganglioside G_M1 (Invitrogen). We followed the product manuals to label lipid rafts before seeding the cells in imaging plates. Briefly, for the Vybrant Alexa Fluor 555 lipid raft labeling kit, we pelleted and washed the cells with prechilled RPMI supplemented with 10% FBS three times and resuspended the cells at the density of 4 × 10⁶ cells per ml. Two microliters of CT-B stock solution (1 mg ml⁻¹) was added to 2 ml of cell solution. The cells were incubated on ice for 15 min and washed twice with prechilled PBS. Thereafter, cells were resuspended in serum-free RPMI and seeded into imaging dishes. After cell adhesion, the cells were fixed with 4% PFA without cell membrane permeabilization and stained with ARPLA. For BODIPY FL C₅-ganglioside G_M1 staining, cell pellets were washed with prechilled HBSS and resuspended in 2 × 10⁶ cells per 100 μl of 5 μM ganglioside G_M1 working solution. The cells were incubated on ice for 30 min, washed with prechilled HBSS three times and resuspended in serum-free RPMI for seeding. After cell adhesion, the cells were fixed and analyzed with ARPLA.

The following antibodies were used to stain SNARE proteins: TSNARE1 polyclonal antibody (Invitrogen), VTI1B polyclonal antibody (Invitrogen) and donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (Invitrogen). HL-60 cells were washed twice with serum-free RPMI, resuspended in serum-free RPMI at a density of 1 × 10⁶ cells per ml and seeded into 35-mm glass-bottom confocal imaging dishes. Cells were then fixed with 4% PFA solution at 37 °C for 15 min and incubated with 0.2% Triton X-100 solution for 5 min at room temperature. After washing with PBS, the cells were stained by ARPLA with minor revisions. Before adding the reporter strand, solutions of primary antibody diluted 1:250 (prepared in 1% BSA) were applied to the cells and incubated at 4 °C overnight. After washing cells with PBS five times, we applied solutions of secondary antibody diluted 1:500 (prepared in 1% BSA) to cells and stained at room temperature for 1 h. We washed cells three times, added ARPLA reporter strand, incubated the cells at 37 °C for 30 min, washed them twice with PBS and mounted the cells in mounting solution.

Fluorescence images were taken with a W1 Nikon spinning-disk confocal microscope or a Zeiss 710 confocal microscope. The images were then processed with Nikon NIS Element viewer or Zen 3.6 (blue version). The colocalization assay was performed with Coloc2 and JACoP plug-ins of Fiji (ImageJ). The plot profiles were also analyzed by using Fiji.