Fluorescence Imaging of Cell Surface Markers

Paired cells on the micropatterned antibody array were first imaged under FLIM mode using a Leica STELLARIS 8 FALCON inverted microscope with each cell’s location recorded. Cells were then washed with PBS and fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS at room temperature for 12 min. After washing with PBS, cells were blocked with 4% HyClone bovine serum albumin (BSA; Cytiva) in PBS at room temperature for 2 hours. Cells were then stained with Alexa Fluor 647–conjugated Tie2 antibody (clone 33, BD Biosciences; 1:100 dilution in 4% BSA) and phycoerythrin-conjugated CD48 antibody (HM48-1, BioLegend; 1:100 dilution in 4% BSA) at room temperature for 2 hours (26 (link)). Imaging of surface markers was carried out on the same microscope under the normal confocal mode.