STORM Assay

For Figure 1B, HEK293T cells were seeded on poly-D-lysine treated coverslips and co-transfected with CD28 and CD80, CD86, or PDL1 fused with an N-terminal SpyTag, using polyethyleneimine. 20 h after transfection, cells were fixed with 4% PFA. CD28 was stained with anti-CD28 antibody (Bio X cell, BE0291) and then with CF568-conjugated anti-mouse IgG antibody (Biotium, 20802). Cells were then incubated with home-made JF646-labeled SpyCatcher protein for staining of Spy-CD80, Spy-CD86, and Spy-PDL1, washed thrice with 1x PBS (pH 7.4), and sealed in Everspark STORM buffer (idylle, Everspark). STORM was performed on a Nikon Eclipse Ti2-E Ti2 inverted microscope equipped with TIRF configuration. The excitation lasers of 561 nm and 640 nm were reflected by a quad dichroic and the emission was filtered by a quad emission filter (open at 581 – 625 nm and 674 – 786 nm). The images were captured using an SR HP APO TIRF 100× 1.49 NA objective (Nikon) and an iXon Ultra 897 EMCCD camera (Andor). CF568 and JF646 fluorophores were converted into a dark state using continuous illumination of excitation light at the maximum intensity. 405 nm laser at low intensity was used to activate fluorophores back to a fluorescent state. Individual blinking CF568 and JF646 fluorophores were recorded for 30,000 to 50,000 frames with 30 milliseconds exposure time. 100 nm Red Fluorescent Nanodiamond (Adámas Nanotechnologies, NDNV100nmHi2ml) was used as a fiducial marker for drift correction. Images were analyzed by ThunderSTORM plug-in⁸⁶ on ImageJ (Fiji), and rendered with an average shifted histogram method. Colocalization between CD28 and B7, or between CD28 and PDL1, was quantified by Manders coefficients using the Coloc-Tesseler software.⁸⁷