Live dead fixable aqua dead stain

Immunofluorescence labelling and flowcytometric analysis was used to identify leukocytes according to cell surface expression of lineage markers as previously described [70 (link)]. In brief, samples of purified leukocytes were labelled with either of the three antibody panels listed in Additional file 5 for 20 min at room temperature in the dark, panel 1 was used on samples both prior and after immunomagnetic separation while panels 2 and 3 were only used with depleted cell samples. LIVE/DEAD® fixable Aqua dead stain (#L34957, ThermoFisher Scientific) was used for dead cell exclusion in all panels. Cells were subsequently washed and fixed with 1.25% paraformaldehyde and stored at 4 °C until analysis.
Flow cytometry was performed using a BD FACSVerse (BD Biosciences), equipped with 488 nm blue, 633 nm red and 405 nm violet lasers and results were analysed using the FACSDiva (BD Biosciences) software. Single-stained compensation controls and fluorescence minus one (FMO) negative controls were included in the assays, the gating strategies are shown in Additional file 6 and samples were recorded until 30,000 events in the CD45 gate were acquired. Titrations of all antibodies were performed to determine optimal labelling conditions prior to the experiment.

4 × 10⁵ sorted naive CD8⁺, CD62L^high, CD44^low T cells from spleens of OT-1 × Arl4d^−/− mice and wild type CD90.1 OT-1 were adoptively transferred in a 1:1 ratio i.v. into CD45.1 recipient mice. One day later mice were infected i.v. with 5 × 10⁶ pfu AdGOL, a recombinant adenovirus expressing GFP, OVA and Luciferase⁴⁷ . After infection 32 μl of blood was taken from the tail-vein at the indicated times and analysed by flow cytometry. Eight days after infection mice were sacrificed and liver lymphocytes and splenocytes were isolated for in vitro analysis. Cells were stained with antibodies against CD45.2, CD45.1, CD90.1, CD8, CD44, CD62L, KLRG1, CD127 and a live/dead stain (Hoechst 33258 (Sigma), near-IR dead cell stain kit or LIVE/DEAD fixable aqua dead stain (Thermo Fischer Scientific)). Fc-block (clone 2.4G2) was added in each staining. To enumerate cells a fixed amount of counting beads was added to the samples prior to acquisition.