Dragonfly spinning disk confocal system

Human Foreskin Fibroblasts (HFF) were purchased from ATCC and cultured in DMEM media (Mediatech) supplemented with 10% Fetal Bovine Serum (Corning), 2 mM L-glutamine (Invitrogen) and penicillin-streptomycin (Invitrogen). HFFs were plated on glass coverslips incubated with 10 μg/mL fibronectin (EMD Millipore) for 1 hr at room temperature. Cells were fixed 1 hr after plating by rinsing them in cytoskeleton buffer (10 mM MES, 3 mM MgCl₂, 1.38 M KCl and 20 mM EGTA) and then fixed, blocked and permeabilized in 4% PFA (Electron Microscopy Sciences), 1.5% BSA (Fisher Scientific), and 0.5% Triton X-100 (Fisher Scientific) in cytoskeleton buffer at 37° for 10 minutes. Coverslips were subsequently rinsed three times in PBS and incubated with either a β₁ antibody (1:100; Abcam product #:ab30394) or β₃ antibody (1:100; Abcam product #:ab7166) followed by AlexaFluor 488 phalloidin (1:1000; Invitrogen) and a AlexaFluor647 donkey anti-mouse secondary antibody (1:200; Invitrogen).
Cells were imaged using a 1.2 NA 60X Plan Apo water immersion lens on an inverted Nikon Ti-Eclipse microscope using an Andor Dragonfly spinning disk confocal system and a Zyla 4.2 sCMOS camera. The microscope was controlled using Andor’s Fusion software.

Th1 migration was assessed under confinement, using an under-agar model as described (41 (link)). Glass coverslips (EMS) were coated with 50 μg/mL FN (Millipore) for 60 min at RT. For pre-treatment of FN, 200 nM FN was incubated with 500 nM pUR4 or III-11C for 30 min at RT before coating coverslips. The coverslip was overlayed with 0.5% agar in serum-free RPMI and 10 ng/mL CXCL10. A small agar plug was then removed to allow ~1 × 10⁵ Th1 cells to be added to the coverslip and slides incubated for 30 min at 37°C to allow for cells to migrate under the agar. For pre-treatment of T cells, 1 × 10⁵ Th1 cells were incubated with 500 nM pUR4 or III-11C for 30 min at 37°C in serum-free RPMI and washed before adding to the imaging chamber. Cells were imaged at 30 s intervals for 20 min using a 20x Plan Fluor oil immersion lens (NA = 0.75) on an inverted motorized Ti-E microscope (Nikon) using DragonFly spinning disk confocal system (Andor).