Fitc conjugated anti cd8a clone 53 6.7

Single-cell suspensions were incubated on ice for 30 minutes with a combination of antibodies specific to cell surface markers for identification lymphocyte subsets. These antibodies are: APC- or PE-Cy7-conjugated anti-NK1.1 (clone PK136), APC-Cy7-conjugated anti-CD3ε (clone 145–2C11), FITC-conjugated anti-CD8a (clone 53–6.7), Alexa Fluor 700-conjugated anti-CD4 (clone RM4–5), PE-conjugated anti-NKG2D (clone CX5), PE-conjugated anti-CD44 (clone IM7), PECy7-conjugated anti-CD25 (clone PC61), FITC-conjugated anti-CD11b (clone M1/70), and/or PE-conjugated anti-Gr1 (clone 1A8) (BD Biosciences). For NK cell receptors, fluorochrome-conjugated NKp46, Ly49A, Ly49C/I/F/H, NKG2A/C/E, CD16 were all from eBiosciences. For intracellular staining, cells were stained with surface markers followed by fixation and permeabilization with BD Perm/Fix kits and antibodies specific to intracellular molecules. Fluorochrome-conjugated antibodies specific to IFN-γ, phospho-AKT (pS473), and phospho-STAT5 (pY694) were all from BD Biosciences. Cells were analyzed using the BD Fortessa. Data were analyzed using the FlowJo software (Tree Star).

Single-cell suspensions were prepared from spleen and lungs as described (21 (link), 45 ). In the case of splenocytes, mice were tested individually. In the case of lung infiltrate cells, cohort analyses were performed with cell pools due to limited cell yield.
Unspecific staining was blocked with unconjugated anti-FcγRII/III antibody (anti-CD16/CD32, clone 93; catalog no. 14-0161; eBioscience, San Diego, CA, USA), and cells were specifically stained with the following antibodies for multi-color cytofluorometric (CFM) analyses: FITC-conjugated anti-CD8a (clone 53-6.7, catalog no. 553031; BD Biosciences, Franklin Lakes, NJ, USA), PE-conjugated anti-KLRG1 (clone 2F1, catalog no. 12-5893; eBioscience), and PE-Cy7-conjugated anti-CD62L (clone MEL-14, catalog no. 731715; Beckman Coulter, Brea, CA, USA). IE1-epitope-specific CD8 T cells were identified by staining with APC-conjugated peptide-folded MHC-I dextramer H-2Ld/YPHFMPTNL (m123/IE1) (Immudex, Copenhagen, Denmark).
A lymphocyte live gate was routinely set in the forward vs. sideward scatter (FSC vs. SSC) plot. All CFM analyses were performed with flow cytometer FC500 and CXP analysis software (Beckman Coulter).