Pe cy7 conjugated anti cd62l clone mel 14

Single-cell suspensions were prepared from spleen and lungs as described above. Unspecific staining was blocked with unconjugated anti-FcγRII/III antibody (anti-CD16/CD32, clone 93; eBioscience, San Diego, CA, USA), and cells were specifically stained with the following antibodies for multi-color cytofluorometric analyses: ECD-conjugated anti-CD8α (clone 53–6.7; Beckman Coulter, Krefeld, Germany), FITC-conjugated anti-KLRG1 (clone 2F1, eBioscience), PE-Cy5-conjugated anti-CD127 (clone A7R34, eBioscience), and PE-Cy7-conjugated anti-CD62L (clone MEL-14, Beckman Coulter). Epitope-specific cells were identified by staining with PE-conjugated, peptide-folded MHC-I dextramers H-2Ld/YPHFMPTNL (m123/IE1), H-2Dd/AGPPRYSRI (m164), and H-2Dd/SGPSRGRII (m18) (Immudex, Copenhagen, Denmark). A lymphocyte live gate was routinely set in the forward vs. sideward scatter plot. Cytofluorometric analyses were performed with flow cytometer FC500 and CXP analysis software (Beckman Coulter).

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).