As 605240

For F-actin polymerization, T cells from Itk^-/-, PI3Kγ^-/-, and WT mice were starved for 1.5 h in a serum-free medium. Where noted, cells were pretreated with 0.5 µM Wortmannin (Calbiochem) for 1 h at 37°C. CCL21 (100 nM; Peprotech) was added to 2 x 10⁶ cells/ml in a 37°C water bath, and cells were fixed at indicated time points in 4% cold PFA. Cells were permeabilized and labeled with FITC-Phalloidin (Invitrogen) as described (19 (link)). For polarization assays, T cells were added on 1 µg/ml fibronectin-coated glass slides before the addition of CCL21 (100 nM) and fixation after 20 min with 2% PFA. After permeabilization with 0.1% Triton X-100 (5 min), cells were incubated with anti-protein kinase ζ; (PKCζ) (H-1; Santa Cruz Biotechnology) and biotin-labeled anti-CD44 (IM7; BD Pharmingen) mAbs. Primary antibodies were detected with a Cy3-labeled anti-mouse Ab (Jackson ImmunoResearch Laboratories) and Alexa488-labeled avidin (Molecular Probes). For chemotaxis assays, T_N was pretreated with inhibitors for DOCK2 (CPYPP) and PI3Kγ (AS-605240; both Tocris) as indicated for 1 h at 37°C, and 0.5 x 10⁶ T_N/well were added to Transwell chambers (5-µm pore size; Costar) to migrate to 50 nM CCL21 for 1.5 h at 37°C in the presence of the inhibitors. Cell viability was assessed by PI staining at the end of the experiment. Input and migrated T cells were enumerated by flow cytometry.

Propidium iodide and DABCO were obtained from Sigma-Aldrich (USA). Quant-iT PicoGreen dsDNA Assay Kit, Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, and PrestoBlue Cell Viability Reagent were acquired from Invitrogen (Thermo Fisher Scientific, USA).
We used the following pharmacological inhibitors/inducers in the study: phorbol 12-myristate 13-acetate (PMA; 100 nM), cytochalasin D (CytD; 10 µg/mL), PD98059 (PD98; MEK inhibitor; 60 µM), and 3-Methyladenine (3-MA; class III PI3K Vps34 and autophagy inhibitor; 5 mM) from Sigma-Aldrich; neutrophil elastase inhibitor III (NEi; MeOSuc-AAPV-CMK; 10 µg/mL), myeloperoxidase inhibitor I (MPOi; 600 nM), and BAPTA-AM (BAPTA; [Ca²⁺]i chelator; 10 µM) from Calbiochem (USA); IC87114 (IC87; selective inhibitor of PI3Kδ; 1 µM) and chloroamidine (Cl-A; PAD inhibitor; 12 μM) from Cayman Chemical (USA); and AS605240 (AS60; selective inhibitor of PI3Kγ; 10 µM) from Tocris Bioscience (UK).
The following antibodies were used in this study: mouse anti-histone H1 monoclonal (sc-8030; 1:100) from Santa Cruz Biotechnology (USA); rabbit anti-myeloperoxidase polyclonal (PA5-16672; 1:200), mouse anti-toxoplasma monoclonal (MA1-83499; 1:100), Goat anti-Mouse Alexa Fluor 488 (A11001; 1:2000), Goat anti-Mouse Alexa Fluor 546 (A11003; 1:400), and Goat anti-Rabbit Alexa Fluor 594 (A11037; 1:800) from Invitrogen.