ADCC was examined using a calcein-acetyoxymethyl (Calcein-AM; Cayman Chemical Co., Ann Arbor, MI, USA) release assay. Canine lymphokine-activated killer (LAK) cells were prepared by culturing canine peripheral blood mononuclear cells from a healthy beagle dog in the presence of 1000 IU/mL of human recombinant interleukin-2 (IL-2) (Novartis, East Hanover, NJ, USA) for 1 week, as previously reported [36 (link)]. CMM2 and KMeC cells were used as target cells. The target cells were labeled with Calcein-AM for 30 min, washed 3 times with PBS containing 5% FBS, and plated onto 96-well plates at a density of 1 × 104 cells/well. P38Bf or whole molecule dog IgG was added at various concentrations from 0.01 to 10 μg/mL for 15 min on ice. Next, the LAK cells were added as effector cells at an effector (E)/target (T) ratio of 10:1. Then, the plates were incubated for 4 h at 37 °C, and the relative light units (RLU) of the supernatants were analyzed using fluorometry to measure calcein release (cell death). For maximal release, the cells were lysed with 2% Triton X-100. Fluorescence was detected using the ARVO X4 system (PerkinElmer, Waltham, MA, USA). ADCC activity was calculated using the following formula:
For each experiment, measurements were conducted in quadruplicate using four replicate wells. Each experiment was repeated at least 3 times.
For each experiment, measurements were conducted in quadruplicate using four replicate wells. Each experiment was repeated at least 3 times.
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