Anti nk

Lymphocytes were isolated from the spleens of each treated mouse, then used for detecting cytotoxic T lymphocyte (CTL) activity [52 (link)]. Briefly, splenocytes isolated from the treated or control mice were depleted of erythrocytes with ammonium chloride Tris buffer. The specific CTL activity was measured by the ⁵¹Cr release assay using CT26 cells as target cells as previously described [53 (link)]. The percent of cytotoxicity was calculated by the formula: cytotoxicity (%) = [(experimental release-effector spontaneous release-Target spontaneous release)/ (Target maximum release-Target spontaneous release)] ×100%. In the cytotoxicity inhibition assays, effectors cells or CT26 cells were incubated with mAb at room temperature for 30 min, washed, and tested. The mAbs included anti-CD8 (10 μg /ml), anti-CD4 (10 μg/ml), anti-NK (10 μg /ml) antibodies (BD PharMingen). The above concentrations of mAb were effective in mediating their activity in preliminary experiments. Control cytotoxicity assay was performed in the presence of isotype IgG mAb (BD PharMingen).

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.