Deltavision elite image restoration microscope

PBMC were stimulated with 1 μg/ml of the HLA-A2-restricted immunodominant peptide CMVpp65_495-503 (NLV) and expanded in a short-term culture for 8d as described above. On d8 CD8⁺ T cells were isolated using a negative CD8⁺ T cell isolation kit (Miltenyi Biotech) according to the manufacturer’s guidelines. T2 cells were pulsed with 1 μg/ml NLV peptide to function as APC.
APC were labelled with Cell Tracker Deep Red (Invitrogen) according to manufacturer’s instructions. CD8⁺ T cells and APC were resuspended in cRPMI with 1% FBS in a 1:1 ratio and incubated on poly-L-lysine coated coverslips for 30 min at 37°C. Cells were then fixed at 4°C in PBS with 4% formaldehyde and 1% bovine serum albumin (BSA) followed by a staining with 10 μg/ml CTB-FITC for 30 min at 4°C in cold PBS with 1% BSA and 0.01% NaN₃. The cells were then permeabilised with 0.1% Triton in PBS with 1% BSA and stained with 10 μg/ml anti-CD3 epsilon mAb (UCHT1, Biolegend) followed by anti-mouse IgG1-AF546 and coverslips mounted with Prolong Gold anti-fade (ThermoFisher). Images were acquired using the DeltaVision ELITE Image Restoration Microscope (Applied Precision) coupled to an inverted Olympus IX71 microscope and a CoolSNAP HQ2 camera, deconvolved with softWoRx 5.0 and processed using Huygens Professional v4.0 and Adobe Photoshop CC 2018.

Quantification of T cell-T cell contacts and VS was performed as described previously (Jolly et al., 2004 (link)). Conjugates were defined as two closely apposed cells consisting of one HIV-1-infected T cell and one target T cell. VSs were defined as conjugates showing enrichment of HIV-1 Env and Gag to the site of cell-cell contact (Jolly et al., 2004 (link), Rudnicka et al., 2009 (link)). Images were acquired using the DeltaVision ELITE Image Restoration Microscope (Applied Precision) coupled to an inverted Olympus IX71 microscope and a CoolSNAP HQ2 camera, deconvolved with softWoRx 5.0 and processed using Huygens Professional v4.0 and Adobe Photoshop C3. Quantification of fluorescence intensity was performed using ImageJ. A region of interest at the contact site (ROI 1) was selected and compared to a region on the opposite side of the cell (ROI 2). The integrated density was adjusted for the size of region of interest and for background, and the fold enrichment of fluorescence signal at the contact zone was determined for at least 20 contacts from two independent experiments.