3 3 dihexyloxacarbocyanine dioc6

The fish were injected with poly(I:C) and dissected to isolate the trunk kidney, spleen, and hepatopancreas as described previously. The cells were isolated by pressing the tissues through a 150-gauge mesh stainless steel sieve in RPMI-1640 medium (Nissui Pharmaceutical). The erythrocytes were lysed with erythrocyte lysis buffer (0.15 M NH₄Cl, 1.0 mM KHCO₃, 0.1 mM EDTA), and the leukocytes were washed twice with PBS.
3,3-Dihexyloxacarbocyanine (DiOC₆, Molecular Probes, Eugene, OR, USA) staining was used to enhance erythrocyte fluorescence and side scatter according to a previously described method for fish^{42 (link)}. The isolated cells were treated with DiOC₆ (10 μg/ml), incubated for 10 min at room temperature, and washed twice with PBS. The lymphocyte and macrophage/granulocytes fractions were gated according to the methods for zebrafish^{20 (link)}.

Both purified CD8⁺ T cells (effector cells) and purified CD4⁺ T cells (target cells) pulsed with 10¹⁰ aldrithiol-2-treated HIV-1 M subtype B isolate (24 (link)) were labeled with 40-nM 3,3′-dihexyloxacarbocyanine (DiOC₆) (25 (link)) (Molecular Probes) for 10 min at 37°C. Target cells were labeled with PerCP-Cy5-conjugated anti-CD4 (BD Bioscience) for 20 min on ice. After washing three times, effector cells were mixed with target cells in a U-bottomed 96-well plate at different effector/target (E/T) ratios (3:1, 1:1, and 0.3:1) in triplicate. K562 cells (target) with APC-conjugated anti-CD32 (BD Bioscience) and purified CD56⁺ (NK) cells (effector) from four HD were included as an assay control. After 4 h incubation at 37°C in the presence of SEB and anti-CD3/anti-CD28, cells were harvested and analyzed by flow cytometry. Percent cytotoxicity was calculated as follows: 100 × (% of total apoptotic target cells − % of spontaneous apoptotic target cells)/(100 − % of spontaneous apoptotic target cells).