Pe cy5 conjugated anti cd127 clone a7r34

Single-cell suspensions were prepared from lung tissue as described (22 (link), 48 (link)). Unspecific staining was blocked with unconjugated anti-FcγRII/III antibody (anti-CD16/CD32; clone 2.4G2, BD Pharmingen, Heidelberg, Germany). 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, Frankfurt), PE-Cy5-conjugated anti-CD127 (clone A7R34; eBioscience, Frankfurt), and PE-Cy7-conjugated anti-CD62L (clone MEL-14; Beckman Coulter). Phenotypic characterization of peptide-specific CD8⁺ T cells was performed using PE-conjugated dextramers H-2Ld/YPHFMPTNL (IE1), H-2Dd/AGPPRYSRI (m164), and H-2Kd/TYWPVVSDI (M105) (22 (link), 31 (link)). H-2Kb/SIINFEKL served as the control for excluding unspecific staining (Immudex, Copenhagen, Denmark). For the analyses, a “live gate” was routinely set on leukocytes in the forward scatter (FSC) versus sideward scatter (SSC) plot. All 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 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).