Sequential expression of intracellular and membrane-associated TNF-α by NK1.1⁺ CD3⁻cells gated from splenocytes.

After 8 hours and 16 hours of incubation in culture medium alone (Ci) or together with anti-Fas mAb (5 μg/mL) plus IFN-γ (100 U/mL) (Cii), intracellular staining of TNF-α was performed according to the manufacturer's instructions (Di and Dii). The membrane-associated form of the cytokine was revealed in the same conditions, except fixation of the cells with PFA and treatment with saponin and brefeldin A. Broken lines represent the isotype control. Intracellular staining of TNF-α in NK1.1⁺CD3⁻ cells (B) gated from freshly isolated NK cells (A) is shown for comparison.