Diff quik staining dye

The phagocytosis was calculated according to (Yoshida, Kitao, 1991) with slight modification, for detail see (Van Doan, Hoseinifar, Sringarm, Jaturasitha, Yuangsoi, Dawood, Esteban, Ringø, Faggio, 2019) . Briefly, 200µL of leucocyte cell suspensions ( 2x 10 6 cells mL -1 ) were loaded on coverslips and incubated at room temperature for two hours. After incubation, the coverslips were washed with 3mL of RPMI-1640 to remove any non-adherent cells. Then, a solution of 200µL of fluorescence latex beads with a concentration of 2 x 10 7 of beads (mL -1 ) (Sigma-Aldrich, USA) was placed into each coverslip and incubated again at room temperature for 1.5 hours. The coverslips were then rewashed with 3mL of RPMI-1640 to remove any non-phagocytized bead. After washing, the coverslips were then fixed with methanol, and stained with Diff-Quik staining dye (Sigma-Aldrich, USA) for ten seconds. After staining, a wash of PBS (pH 7.4) removed any excessive stains. The washed coverslips were allowed to dry at room temperature and then attached to the slides with Permount (Merck, Germany). The number of phagocyte cells per 300 adhered cells was later counted microscopically. The phagocytic index (PI) and phagocytic rate (PR%) were calculated through the following equations: PI = (Number of phagocytized beads divided by the number of phagocytizing leukocytes) *100.

Phagocytosis activity was measured via the procedure specified in Yoshida and Kitao [102] . Briefly, 200µL of leucocyte cell suspensions (2 x 10 6 cells mL -1 ) were loaded on coverslips and incubated at room temperature for two hours. After incubation, the coverslips were washed with 3mL of RPMI-1640 to remove any non-adherent cells.
Then, a solution of 200µL of fluorescence latex beads with a concentration of 2 × 10 7 of beads (mL -1 ) (Sigma-Aldrich, USA) was placed into each coverslip and incubated again at room temperature for 1.5 hours. The coverslips were then rewashed with 3mL of RPMI-1640 to remove any non-phagocytized bead. After washing, the coverslips were then fixed with methanol, and stained with Diff-Quik staining dye (Sigma-Aldrich, USA) for ten seconds. After staining, a wash of PBS (pH 7.