Abstract
<p>This protocol describes the preparation and single-cell spatial imaging of adherent cultured cells on the Bruker/NanoString CosMx SMI platform using the Human 6K Discovery Panel. It adapts the standard CosMx SMI RNA fresh-frozen manual slide-preparation workflow (Bruker Spatial Biology, MAN-10184) for paraformaldehyde (PFA)-fixed cells grown directly on CosMx-compatible glass slides inside removable silicone chambers. The chamber format enables spatially segregated growth of multiple samples on shared slides; here, two donors and four timepoints across a fibroblast conversion timecourse. Modifications relative to the standard tissue workflow include a photobleaching step, omission of heat target retrieval, a gentler Proteinase K permeabilization, adjusted nuclear and antibody segmentation-marker concentrations, a second acetylation, and a second fiducial application. These changes are designed for the fragility of cultured cells and minimizing common CosMx errors.</p>