Facs canto 1 cytometer

Cells were seeded in 24-well plate (1 x 10⁵ cells/well) and pre-treated (when required) for 3 h with MRT67-307 inhibitor (Sigma, SML0702) used at a concentration from 5 μM to 25 μM. Transduction was performed using 50 pp/cell of WT or M1 vectors expressing either GFP or mCherry, in the presence or absence of inhibitor. Three hours post infection, complete DMEM containing virus was removed and replaced by complete DMEM overnight at 37°C. Twenty-four hours later, cells were washed in PBS and collected using 200 μL of trypsin and 300 μL of PBS with 0.6 mM EDTA and analyzed by flow cytometry for GFP and/or mCherry expression using FACS Canto I cytometer (BD Biosciences). For each condition, 10000 cells were analyzed using FACS DIVA software.

Supernatants obtained from fresh lung suspensions (total volume 3 mL) were aliquoted and frozen at −80°C. The aliquots were thawed (only once) and the cytokines present in a 25 μL sample were analyzed (CXCL1, IL-18, IL-23, IL-12p70, IL-6, TNF-α, IL-1β, and IL-12p40) with the LEGENDplex Cytokine Analysis kit (BioLegend), according to the manufacturer’s recommendations, and using a FACS Canto I cytometer (BD Biosciences) and the BioLegend LEGENDplex software.

On day 7 to 14 after primary stimulation, reactivity of TCL was examined by flow cytometry. Cells were washed three times and incubated in the presence or absence of 10 µg/ml abacavir for 6 h with CFSE labelled autologous PBMC. After 2 h of co-incubation Brefeldin A (10 µg/ml, Sigma-Chemicals, Buchs, Switzerland), Monensin (6 µg/ml, Sigma-Chemicals, Buchs, Switzerland) and anti-CD107a-PE antibody (Biolegend, San Diego, CA, USA) were added. Surface staining was performed with anti-CD3-PerCp-Cy5.5, anti-CD4-PE-Cy7 and anti-CD8-APC-Cy7 (Biolegend, San Diego, California). Intracellular staining with anti-IFNγ-APC was performed according to the Cytofix/Cytoperm permeabilization kit (BD Biosciences, Basel, Switzerland). FACS analysis was performed on a FACSCanto-I cytometer using FACS-Diva software (BD Biosciences).