Ultraview spinning disc confocal microscope

To assess internalization of target/antibody complex, HER2-expressing SKBR-3 cells were treated with pHrodo™ Red (acidic pH fluorescent indicator) directly conjugated test antibodies for a period of 4 h at 37°C. Control samples were incubated with test antibodies at 4°C to prevent internalization. Two orthogonal approaches were used to observe internalization: flow cytometry, allowing a per cell distribution analysis of pHrodo Red signal, and immunofluorescence confocal microscopy allowing for a qualitative visualization of receptor internalization. Flow cytometric acquisition was performed on an LSRII (Becton Dickinson, San Jose, CA), and analysis was performed using FlowJo flow cytometry analysis software, version 10.4 (Tree Star, Inc., Ashland, OR, USA). Confocal imaging was performed with an Ultraview spinning-disc confocal microscope (PerkinElmer, Shelton, CT, USA) using Volocity image acquisition/analysis software, version 6.3 (PerkinElmer, Shelton, CT, USA).

Cultures were processed for immunocytochemistry as previously described (Brizuela et al., 2015 (link)). Primary antibodies (rat anti-GFP, 1:3,000, Nacalai tesque, RRID: AB_10013361). Secondary antibodies (Alexa Fluor anti-rat 488, 1:1,000, Molecular Probes, RRID: AB_2534074). Neurobiotin-filled interneurons were labeled with streptavidin-546 and imaged using a UltraView Spinning disc confocal microscope (Perkin Elmer) with Velocity Software (Velocity v6 3.0, 2013, Perkin Elmer).