Flow cytometric quantification of lung natural killer cell activity associated with TLR-3 signalling pathway activation


  1. Dai, X.
  2. McLaws, L.J.
  3. Schnell, G.J.
  4. Viswanathan, S.
  5. Hu, C.C.
  6. Bhogal, H.S.
  7. Wong, J.P.
Corporate Authors
Defence R&D Canada - Suffield, Ralston ALTA (CAN)
The objective of this study was to evaluate natural killer (NK) cell activation as a potential immunological biomarker for antiviral protection provided by pretreatment with liposome-encapsulated (LE) Poly ICLC, a toll-like receptor-3 agonist. To achieve this, lungs of mice intranasally treated with LE Poly ICLC or free Poly ICLC (1 mg/kg body weight) were aseptically harvested at various time points post drug treatment and NK cells were then isolated and used as effector cells in cytotoxicity assay. A flow cytometric assay using dual fluorescent dyes was developed to examine the duration of lung-associated NK cytotoxic activity in mice. Two methods of flow cytometric analysis were employed: one based on 7-amino-actinomycin D (7-AAD) viability dye incorporation and the other combining 7-AAD incorporation with forward scatter (FSC) parameters. The results from both methods demonstrated that LE PolyICLC and free PolyICLC-induced NK cytotoxicity was significantly higher than the basal levels from the PBS-treated lungs at 7 days post intranasal administration (p < 0.05). However, LE PolyICLC-augmented cytotoxicity remained elevated through 14 days, significantly higher than the basal levels from the PBS-treated lungs. In comparison, PolyICLC-induced cytotoxicity gradually declined at 14 days (p > 0.05 vs PBS control). Our results demonstrated that LE Poly ICLC induced a potent augmentation of lung-associated NK activity for 14 days post intranasal treatment. The correlation betwe

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Report Number
DRDC-SUFFIELD-TM-2010-188 — Technical Memorandum
Date of publication
01 Nov 2010
Number of Pages
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