Flow Cytometry Analysis of Flounder Leukocytes

Leukocytes (1 × 10⁶ cell/mL) from kidney, liver, spleen, and PBL from three olive flounders (62.3 ± 9.6 g at 16 °C) were prepared by centrifugation at 500 × g for 3 min according to Section 2.6. For flow cytometry analysis, leukocytes were incubated with respective CD marker mAbs- 4B2 (anti-CD3), 10F8-3 (anti-CD4-1) and 1D3 (anti-CD4-2) for 1 h at RT (room temperature, 25 °C), respectively [26 (link),27 (link),28 (link)]. This was followed by fluorescein isothiocyanate (FITC)-conjugated AffiniPure goat antimouse IgG (H + L; Jackson ImmunoResearch, Pennsylvania, USA) staining with a 1:200 dilution for 30 min at 37 °C. Negative controls were treated with FITC conjugate only. Leukocytes were washed twice with 1× PBS between each step, re-suspended in 1× PBS, and analyzed using a FACS Calibur™ (BD biosciences, San Jose, USA) flow cytometer, measuring at least 10,000 events per sample.