Immunofluorescence labelling for flow cytometry

T cells were plated with sAg-pulsed CP50-EBV cells, previously loaded for 15 min with 0.5 μM 5-chloromethylfluorescein diacetate CellTracker green CMFDA (Molecular Probes–Eugene, OR, USA) at ratio 1:1. Cells were conjugated by 1 min centrifugation. After 5 h of coculture, TCR internalization was measured by extracellular staining of the CD3ɛ subunit with AlexaFluor 647-coupled anti-CD3ɛ (clone UCHT1, BD Pharmingen). CD69 was stained with a PE-coupled anti-CD69 mAb (clone L78, BD Pharmingen). For cytokine production assays, brefeldin A (5 μg ml⁻¹; Sigma-Aldrich) was added after the first hour of coculture. After an additional 3–18 h of incubation, cells were fixed in 1% PFA, permeabilized with 0.1% saponin and stained with PE-coupled anti- IFN-γ (clone B27), APC-coupled anti-IL-2 (clone MQ1-17H12) and AlexaFluor 700-coupled anti-TNFα (clone MAb11; all from BD Pharmingen). Cells were acquired on a FACSFortessa (BD Biosciences–San Jose, CA, USA). CD3ɛ surface expression, IFN-γ or IL-2 or TNFα-positive subset were estimated for the CMFDA-negative population, using software FlowJo. For the detection of the high-affinity form of LFA-1, T cells were stained with 0.5 μM CMFDA and stimulated with sAg-pulsed B cells in presence of antibody 24, which is specific for the high-affinity conformation of LFA-1 (2 μg ml⁻¹, clone 24, Abcam–Cambridge, UK). After 25 min of coculture, cells were washed, fixed and stained with an AlexaFluor647-coupled secondary antibody. High-affinity LFA-1 staining was estimated for CMFDA-positive cells. Data were acquired on a FACSFortessa and analysed using software FlowJo.