Anti ifn γ clone xmg1

Spleens were collected and tissue was processed into single-cell suspension. Surface staining was conducted by incubating splenocytes with appropriate antibody cocktails for 20 min at 4°C. Endogenous memory and naïve CD8 T cells were detected based upon surface staining with anti-CD8 (clone 53-6.7, eBioscience) and anti-CD11a (clone M17/4, eBioscience) as previously described (30 (link)). P14 cells were detected based upon surface staining with anti-CD8 and anti-Thy1.1 (clone His51, eBioscience), and in some instances 1° earlyM and lateM or 1° and 3° memory P14 cells were distinguished from one another based upon additional surface staining with anti-Thy1.2 (clone 53-2.1, eBioscience). Intracellular cytokine staining was performed using anti-IFN-γ (clone XMG1.2, eBioscience) or anti-granzymeB (anti-GrB; clone GB12, Invitrogen). Flow cytometry data was acquired using FACSCanto (BD Biosciences, San Jose, CA, USA) and analyzed using FloJo software (Tree Star Inc., Ashland, OR, USA).

Spleens were isolated from H37Rv- and H37RvΔTlyA-infected mice and macerated by frosted slides in 10% RPMI 1640 medium (Gibco, Invitrogen) and made into a single cell suspension. RBCs were lysed with RBC lysis buffer, incubated at room temperature for 2–3 min, and washed with 10% RPMI 1640 medium. The cells were counted, and 1 × 10⁶ cells were used for surface staining. Cells were harvested and washed twice with PBS and stained with fluorescent antibodies directed against surface markers. For intracellular staining, 1 × 10⁶ cells were cultured per well in 24-well plates (Corning, Co-star) and activated with 50 ng/ml phorbol 12-myristate 13-acetate (Sigma) and 750 ng/ml ionomycin (Sigma) overnight, and 10 mg/ml brefeldin A (eBiosciences) was added during the last 3 h of culture. After staining, cells were washed again with PBS, and cells were fixed with 100 μl of fixation buffer (eBiosciences) for 15 min, then resuspended in 200 μl of 1× permeabilization buffer (eBiosciences), and stained with fluorescently conjugated monoclonal antibodies as follows: anti-IL-4 (clone, 8D4-8), anti-IL-17 (clone, 17B7), and anti-IFN-γ (clone, XMG1.2), all from Pharmingen. Fluorescence intensity of fluorochrome-labeled cells was acquired and analyzed by flow cytometry (FACS Canto II, BD Biosciences). Data analysis was performed by Flow Jo (Tree Star).