Perforin and granzyme B staining of NK cells

PBMCs (2 × 10⁵ cells) were incubated with different concentrations of PGD₂ (10 or 25 ng/mL) for 5 h at 37 °C. The cells were then stained for surface markers with anti–CD3-PerCP and anti–CD56-PE antibodies. After washing the PBMCs twice with FACS buffer, they were incubated in BD Cytofix/Cytoperm solution. Before and after intracellular staining with anti–perforin-Alexa647 and anti-granzyme B-Alexa 647, the cells were washed twice with BD Perm/Wash buffer and then analyzed by flow cytometry gated on CD3-CD56+ NK cells.