Figure 1
From: Validation of a Miniaturized Permeability Assay Compatible with CRISPR-Mediated Genome-Wide Screen
Scheme of the MAP design. (a) Gelatin-coated MCs composed of cross-linked dextran carry a confluent EC monolayer (green, to designate calcein-loaded ECs), incubated in medium containing the collagen-binding proteolytic fragment of fibronectin conjugated to a fluorophore (FNcf). Once treated by a junction-disrupting agonist (thrombin, in this study), FNcf binds to the exposed gelatin surface between cells whose junctions disassembled in response to the agonist. (b) MCs carrying untreated ECs bind a minimal amount of FNcf. MCs carrying agonist-treated ECs bind varying amounts of FNcf,, depending on the identity of the sgRNA expressed by the clonal cell population on each MC. MCs carrying ECs that express sgRNAs targeting genes that encode proteins required for the induction of the disassembly of cell-cell junctions bind a low amount FNcf,, similar to untreated MCs. ECs expressing sgRNAs that are unrelated to the signaling pathway of the junction-disrupting agonist respond by disassembling their junctions. FNcf binds to the gelatin surface exposed between the responsive ECs, rendering the MCs that carry these cells fluorescent. The fluorescent MCs are separated from the dark MCs by fluorescence-assisted sorting. The gates of the sorting machine can be set up to capture any group of interest in this population, based on MC fluorescence amplitude.
