Anti nkg2a

Cells were stained with saturating amounts of various fluorescent-labeled antibody combinations including anti-EpCAM (EBA-1 clone), anti-CD45 (HI30 clone), anti-CD3e (UCHT1 clone), anti-CD56 (B159 clone), anti-CD4 (SK3 clone), anti-CD8 (SK1 clone), anti-CD25 (M-A215 clone), anti-CD107a (H4A3 clone), anti-CD45RO (REA611 clone), anti-NKG2A (REA110 clone), anti-NKG2D (BAT221 clone), anti-CD137 (4B4–1 clone), anti-CD16 (3G8 clone), anti-HLA-E (3D12 clone), Annexin V and co-stained with DAPI (all from BD Biosciences or Miltenyi). Cells were analyzed with the Attune NxT flow cytometer (Life Technologies) and further analyses were performed with FlowJo software (Tree Star).

pDCs were harvested after activation with IMQ and CpG for 48 hours, and stained with the following mAbs: anti-CD11c, anti-CD11b, anti-B220, anti-CD80, anti-CD86, anti-MHC II (BD Pharmingen, USA), anti-TRAIL, and anti-Granzyme B (eBioscience, USA). Mice were injected i.t. with resting or activated pDCs, and sacrificed on day 2 or 5. Single-cell suspensions were prepared from tumor tissues. After enzymatic digestion for 30 minutes at 37°C with type IA collagenase (1 mg/mL) and DNase (0.1 mg/mL), red blood cells were lysed with Pharmlyse Buffer (BD Biosciences, USA); and stained with the following mAbs: anti-CD45, anti-NK, anti-CD3, anti-CD8, anti-TRAIL, anti-NKG2D, anti-NKG2A, and CD107 (BD Pharmingen, USA). Intracellular staining was performed using a cell permeabilization kit (Fix & Perm; An Der Grub). After incubation with the respective Abs for 20 minutes at 4°C, cells were washed twice and subjected to flow cytometric analysis. FACS plots depict the mean fluorescence intensity (MFI) values of Ab staining after subtraction of the MFI of the respective isotype.