Abstract
Intracellular protein delivery is a transformative tool for biologics research and medicine. Delivery into the cytosol allows proteins to diffuse throughout the cell and access subcellular organelles. Inefficient delivery caused by endosomal entrapment is often misidentified as cytosolic delivery. This inaccuracy muddles what should be a key checkpoint in assessing delivery efficiency. Green fluorescent protein (GFP) is a robust cargo small enough to passively diffuse from the cytosol into the nucleus. Fluorescence of GFP in the nucleus is a direct readout for cytosolic access and effective delivery. Here, we highlight recent examples from the literature for the accurate assessment of cytosolic protein delivery using GFP fluorescence in the cytosol and nucleus.