Anti cd3 allophycocyanin

MNCs were stained with anti-CD3 allophycocyanin (APC) and anti-CD56 fluorescein isothiocyanate- (FITC-) conjugated mouse anti-human monoclonal antibodies, from Becton-Dickinson (BD Pharmingen, San Diego, CA, USA) as previously described [4 (link)]. Intracellular perforin and granzyme B expressions in NK cells were investigated after permeabilization of the cell membrane with a Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA, USA) using PE or PerCP-conjugated anti-perforin or anti-granzyme B (BD Pharmingen, San Diego, CA, USA), according to the manufacturers' instructions.
The fluorescent staining was analyzed on a FACSCalibur (BD Biosciences) flow cytometer. Appropriate fluorochrome-conjugated isotype-matched controls were used for background staining. Each analysis was performed using at least 10,000 cells that were gated in the viable lymphocyte population, as determined by forward scatter versus side scatter properties. The lymphocytes were gated to identify CD3⁺and CD3⁻ cells for further analysis of the expression levels of CD56. The NK cells were defined as CD3⁻ and CD56⁺. As shown in Figure 1(a), the two subpopulations of NK cells, CD56^dim and CD56^bright NK subsets, could be further defined according to the density of expression of CD56.

The following fluorescence-labeled monoclonal antibodies against cell-surface antigens were used: fluorescein isothiocyanate-anti-CD107a, peridinin chlorophyll-Cy5.5-anti-NK1.1, and allophycocyanin-anti-CD3 were purchased from BD Biosciences (San Jose, CA, USA); phycoerythrin (PE)-CY7-anti-IFN-γ was purchased from eBioscience (San Diego, CA, USA); anti-PE microbeads were purchased from Miltenyi Biotec (Bergisch Gladbach, NRW, Germany); and the PE-anti-CD1d tetramer was obtained from the National Institutes of Health Tetramer Core Facility (Atlanta, GA, USA). The cells were stained with the indicated antibodies and analyzed using an FACSCalibur flow cytometer (BD Biosciences, NJ, USA) and FlowJo 7.6.1 software.