Anti cd56 apc

The use of human PBMCs, including NK cells from healthy donors, was approved by the local ethical committee (#EK242102007) of the Medical Faculty Carl Gustav Carus, Technische Universitat Dresden. After obtaining oral and written consent, PBMCs were isolated by Biocoll gradient centrifugation (Biochrom, Berlin, Germany) and human cytomegalovirus (HCMV) IgG and IgM titers were analyzed using ELISA. Untouched NK cells were prepared using the negative NK Cell Isolation Kit (Miltenyi Biotec). Staining with anti-CD56-APC, anti-CD3-FITC and anti-CD19-PE antibodies (Miltenyi Biotec) routinely confirmed 90% purity of CD56 + NK cells and depletion of CD3 + T cells and CD19 + B cells. B cells were isolated from PBMCs using the negative Pan B Cell Isolation Kit (Miltenyi Biotec). PBMCs and NK cells were held at 37°C and 5% CO 2 in a humidified incubator.

Apoptosis was analysed using annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) as previously described [17] . Briefly, following treatment cells were washed with annexin V binding buffer (5 mM HEPES, 70 mM NaCl, 1.25 mM CaCl 2 pH 7.4) and stained with annexin V-FITC (iQ Corporation, Groningen, The Netherlands). Following washing with annexin V binding buffer, cells were resuspended in PI (0.5 μg/mL) in binding buffer and analysed on either a BD Accuri C6 or BD FACS Canto II flow cytometers (BD Sciences) using FloJo software (Ashland, Oregon, United States).
To determine the cytotoxic effects of VP79s in lymphocytes, 1 × 10 5 normal donor PBMCs per well were treated as indicated and stained with anti-CD56-APC (NK cells), anti-CD19-PE (B cells) and anti-CD3-PerCP (T cells) antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) as per manufacturer's protocol. Samples were analysed by flow cytometry on the BD FACS Canto II flow cytometer with cell viability determined by DAPI exclusion (Pacific blue).
Cell cycle was analysed by DNA quantification following staining by propidium iodide (PI). After treatment, NCI-H929 and U266B1 cells were fixed in 70% ethanol and incubated with 10 μg/mL of RNase A (Sigma-Aldrich) and 100 μg/mL PI (Sigma-Aldrich). Analysis was performed using a BD Accuri C6 flow cytometer and software.