Anti brdu

For synchronization experiments, 200 000 cells/ml were plated in a six-well dish. The next day thymidine (2 mM final) was added to the cells, 18 h later the cells were washed three times with PBS and complete medium was added for 9 h. Then thymidine (2 mM final) was added for 18 h. The cells were then washed three times in PBS and complete medium containing BrdU was added. For BrdU cell cycle analysis of non-synchronized cells, 100 000 cells per well were plated. The next day BrdU was added to the plate for 20–30 min. The cells were harvested and washed in PBS then fixed in 70% ethanol overnight at 4 °C. The cells were treated with 3N HCl that was neutralized with 0.1 M Na2B4O7 pH 8.5. The cells were then blocked and stained with an anti-BrdU (Biolegend, London, UK) and 7-AAD (Life Technologies). For apoptosis experiment, Apoptosis detection kit I (Becton Dickinson, Le Pont de Claix, France) was used following manufacturer protocol. For TLR9 intracellular staining, one million cells were fixed with 2% paraformaldehyde and permeabilized in PBS containing 0.25% saponin. After blocking the cells were stained with a rat anti-human TLR9 (eBioscience, Paris, France) and with a secondary goat anti-rat Alexa 633. The staining was analyzed on a BD LSRII using the software Diva and FlowJo (Treestar, Ashland, OR, USA).

We used rabbit anti-ESAT-6 antibody (Abcam, Cambridge, MA), mouse anti-His_6X antibody (Sigma-Aldrich, St. Louis MA), anti-FLAG antibody (Sigma-Aldrich, St. Louis MA), HRP conjugated anti-mouse IgG (Bio-rad, Hercules, CA), anti-rabbit FITC conjugated secondary antibody (Jackson-ImmunoResearch, West Grove, PA) and anti-mouse Texas Red secondary antibody (Jackson-ImmunoResearch, West Grove, PA) for in vitro studies. For in vivo studies, anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5 or -APC, anti-CD8 (clone: 53–6.7)-FITC, -PerCP-Cy5 or -APC, anti-CD44 (clone: IM7)-APC, anti-Brdu (clone: Bu20a)-PE, anti-CD11b (clone: M1/70)-APC, anti-CD11c (clone: N418)-APC, 7AAD, anti-IFN-γ (clone: XMG1.2)-APC, anti-IL-17 (clone: TC11-18H10.1)-PE, anti-IL-4 (clone: 11B11)-PE, anti-IL-6 (clone: MPS-20 F3)-PE, anti-IL-12 (clone: C15.6)-PE, anti-IL-22 (clone: Poly5164)-PE, anti-IL-10 (clone: JES5-16E3)-PE, anti-IL-9 (clone: MH9A4)-PE, anti-TNF-α (clone: MP6-XT22)-PE, (all from Biolegend, USA) and anti-CD69 (clone: H1.2 F3)-PE (from eBiosciences, USA) were used.

We used the following antibodies: anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5, or -APC, anti-CD8 (clone: 53-6.7)-FITC, -PerCP-Cy5, or -APC, anti-CD44 (clone: IM7)-APC, anti-BrdU (clone: Bu20a)-PE, 7AAD, anti-IFN-γ (clone: XMG1.2)-APC, anti-IL-17 (clone: TC11-18H10.1)-PE, anti-IL-4 (clone: 11B11)-PE, anti-IL-6 (clone: MPS-20F3)-PE, anti-IL-12 (clone: C15.6)-PE, anti-IL-22 (clone: Poly5164)-PE, anti-IL-10 (clone: JES5-16E3)-PE, anti-IL-9 (clone: MH9A4)-PE, anti-TNF-α (clone: MP6-XT22)-PE (all from Biolegend, USA), anti-Active Caspase-3 (clone: C92-605)-FITC or -PE (from BD Pharmingen™, USA), and anti-CD69 (clone: H1.2F3)-PE (from eBiosciences, USA).