Volocity imaging analysis software

For translocation analysis, Jurkat T cells expressing wt-IG4_GFP or other mutants were incubated with SEE-pulsed Raji B cells stained with CellTracker orange CMRA (Invitrogen) for 30 min and placed on PLL-coated coverslips or anti-CD3-antibody-coated coverslip for 30 min. The accumulation of wt-IG4_GFP or other mutants at the T cell-APC contact site or anti-CD3-coated surface was calculated as the ratio of fluorescence intensity at the contact region (Fcon = c) to the fluorescence intensity at the opposite site (Foppo = o). Fluorescence intensity was represented by an intensity profile, with blue indicating the lowest intensity and red indicating the highest intensity. To evaluate CD3 ϛ−clustering in MT4 cells, scrambled or IGSF4 siRNA-treated cells were transfected with CD3 ϛ_GFP using Amaxa. The cells were then placed on PLL at 24 h post-transfection and imaged using a 100×, NA 1.40 oil immersion objective lens on a laser-scanning confocal microscope (FV1000; Olympus, Tokyo, Japan). CD3 ϛ_GFP clustering was identified using the “find objects using intensity” and “separate touching objects (object size guide 0.2 μm²) functions of Volocity imaging analysis software (PerkinElmer).

To measure the number and area of TCR microclusters, EV- or IG4ΔEXT-OTI CD8⁺ T cells were stained with TCRβ (H57Fab-594). The cells were then placed on a planar lipid bilayer with OVA257-265-H-2K^b/ICAM-1 and immediately imaged for 20 min by TIRFM (IX-81; Olympus, Tokyo, Japan) equipped with a solid-state laser (488 nm, 20 mW; Coherent, Santa Clara, CA, USA). The generation of bilayers has been described previously (29 (link), 30 (link)). Microclusters were identified using the “find objects using intensity” and “separate touching objects (object size guide 0.2 μm²) functions of Volocity imaging analysis software (PerkinElmer). In this analysis of lipid bilayer imaging, only the clusters generated within 1 min after attachment were analyzed.