Expression analysis and co-localization imaging of Jump In T-REx HEK293 WT-OE cell pools

The Method uses HEK293TrexR4 cells (=wt) cells and genetically engineered variants (wt OE, KD, other) to provide a basis for expression analysis and to detect the localization of SLC-HA-Strep- tag in cellular compartments. This protocol describes the physical preparation of microscopic samples. Image analysis with respect to compartmental co-localization of the SLCs respectively their detection tags will be covered separately.

Therefore, we describe how an HA Tag is co-stained with Hoechst 33342 and specific compartment markers for Golgi, Lysosomes, ER, Mitochondria, Cell Membrane, Peroxisomes, Endosomes and Cytoplasm in order to either measure the total intensity of expressed SLC-HA tag protein per cell or give estimates on their cellular distribution. We chose a 60x magnification using water immersion objectives and a well bottom with 192um COC film thickness to provide high quality images. For compatibility reasons also 20x images are recorded using HCS cell mask stains as a backdrop to find cytoplasmic outlines.