Lysobrite blue

Fluorescence labeling of the proteins was performed according to the AlexaFluor568 carboxylic acid NHS ester manufacturer’s instructions (ThermoFisher Scientific (Waltham, MA, USA)). SKBR-3-Luc (JCRB1627.1), MCF-7-Luc (JCRB1372), and HCC-1954-Luc (JCRB1476) were purchased from JCRB (Tokyo, Japan). For internalization 5000 cells per well were seeded in a µ-slide 8-well chamber coverslip (ibidi (Gräfeling, Germany)) and incubated for 48 h at 37 °C, 5% CO₂ (RPMI1640, 10% (v/v) fetal bovine serum (FBS), ThermoFisher Scientific (Waltham, MA, USA)). The medium was replaced by FluoroBrite DMEM medium with 10% (v/v) FBS (ThermoFisher Scientific (Waltham, MA, USA)) and 50 nM labeled Fab or bsFab was added and incubated for 24 h. Before starting microscopy, cells were washed with FluoroBrite DMEM medium without FBS. Cells were mixed with equivalent amounts of FluoroBrite DMEM medium and life cell staining buffer (supplemented with either LysoBriteBlue (1:500), AATBioquest (Sunnyvale, CA, USA), or HOECHST33342 (1:2000), ThermoFisher Scientific (Waltham, MA, USA)) and incubated prior to microscopy for 0.5–2 h and 10 min, respectively (Eclipse TE2000-E, CFI Plan Fluor 40x oil lens, Nikon (Tokyo, Japan)). Lysosome and nucleus staining were excited at 405 nm (filter 450/35), fluorescent proteins at 568 nm (filter 650LP).