Multiparametric Analysis of T Cell Responses

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).