Anti–HA-ATTO-565 and Tf-ATTO-565 were generated by labeling anti-HA antibodies (Sigma-Aldrich, #901501) and human Tf (Sigma-Aldrich, #616397) with ATTO-565-AEDP-NHS-ester (Atto-tec, Siegen, Germany). For anti-HA antibody conjugation, ATTO-565-AEDP-NHS-ester was added to anti-HA antibodies such that the molar ratio of dye to antibody was approximately 10:1. The mixture was incubated at room temperature (RT) for 1 hour. For Tf conjugation, ATTO-565-AEDP-NHS-ester was added to Tf (5 mg/ml) such that the molar ratio of dye to Tf was approximately 10:1. The mixture was incubated at RT for 1 hour, followed by incubation for 18 hours at 4°C. After centrifugation to remove precipitates, the samples were transferred to dialysis tubing and dialyzed for 24 hours at 4°C against 1 liter of PBS to remove free dyes. Protein concentrations were measured using Coomassie protein assay reagents (Thermo Fisher Scientific, #1856209). After the addition of the preservative Kathon CG/ICP (Supelco Analytical, #5-00119), the samples were stored at 4°C protected from light.
On the day of endocytosis assays, cells were washed three times with KRH buffer and starved in KRH buffer for 30 min before being chilled on ice. Subsequently, the cells were incubated with prewarmed HA-ATTO-565 (1 μg/ml) in the presence of 20 nM insulin or Tf-ATTO-565 (5 μg/ml) in a 37°C incubator for desired durations. The cells were chilled on ice and washed three times with 150 mM ice-cold MESNa (Sigma-Aldrich, #M1511) in PBS and once with ice-cold PBS. Subsequently, the cells were disassociated using Accutase, and ATTO-565 fluorescence was measured using the Phycoerythrin (PE) channel (filter of 586/15 nm) on a MACSQuant Analyzer. About 10,000 cells of each sample were analyzed. To detect endocytosis by confocal imaging, cells were grown on micro cover glasses with a diameter of 12 mm (VWR, #89015-725) in 24-well plates. Endocytosis assays were carried out as described above. After washing with MESNa three times and once with PBS, the cells were fixed with 4% PFA. After washing three times with PBS, the cells were incubated with CF405M-conjugated concanavalin A (50 μg/ml; Biotium, #29074) for 20 min at RT to stain the cell membrane. The 405-nm laser was used to detect CF405M-conjugated concanavalin A (cell membrane staining), and the 561-nm laser was used to detect ATTO-565 fluorescence using a 100× oil immerse objective on a Nikon A1 laser-scanning confocal microscope. Tf-ATTO-565–positive endocytic vesicles were analyzed using Fiji.
On the day of endocytosis assays, cells were washed three times with KRH buffer and starved in KRH buffer for 30 min before being chilled on ice. Subsequently, the cells were incubated with prewarmed HA-ATTO-565 (1 μg/ml) in the presence of 20 nM insulin or Tf-ATTO-565 (5 μg/ml) in a 37°C incubator for desired durations. The cells were chilled on ice and washed three times with 150 mM ice-cold MESNa (Sigma-Aldrich, #M1511) in PBS and once with ice-cold PBS. Subsequently, the cells were disassociated using Accutase, and ATTO-565 fluorescence was measured using the Phycoerythrin (PE) channel (filter of 586/15 nm) on a MACSQuant Analyzer. About 10,000 cells of each sample were analyzed. To detect endocytosis by confocal imaging, cells were grown on micro cover glasses with a diameter of 12 mm (VWR, #89015-725) in 24-well plates. Endocytosis assays were carried out as described above. After washing with MESNa three times and once with PBS, the cells were fixed with 4% PFA. After washing three times with PBS, the cells were incubated with CF405M-conjugated concanavalin A (50 μg/ml; Biotium, #29074) for 20 min at RT to stain the cell membrane. The 405-nm laser was used to detect CF405M-conjugated concanavalin A (cell membrane staining), and the 561-nm laser was used to detect ATTO-565 fluorescence using a 100× oil immerse objective on a Nikon A1 laser-scanning confocal microscope. Tf-ATTO-565–positive endocytic vesicles were analyzed using Fiji.
