Figure 3. Identification of MH2 as macrophage-like phagocytic cells.
(A) Comparison between MH2 and human macrophage marker genes. (B) A representative contour plot of shrimp hemocytes against FITC-VP. Threshold intensity (FITC-A) was set to <103 representing control hemocytes (R2), and >2 × 103 representing phagocytic hemocytes (R1). R1 and R2 were sorted based on the forward scatter (FSC) and fluorescence intensity (FITC) two-dimensional space. (C) Confocal microscopy of sorted hemocytes (R1) with ingested FITC-labelled Vibrio Parahemolyticus. Green, ingested Vibrio Parahemolyticus; Blue, nuclei. Scale bar: 10 μM. (D) Efficiency of the phagocytosis inhibitor on the Vibrio Parahemolyticus uptake of shrimp hemocytes. The results are presented as mean ± SD of 6–8 replicates. Asterisks denote statistical significance (**p=0.007) between the control and different treatments. (E) Differential gene expression analysis (CHIT1 (**p=0.004), Lyz1 (*p=0.049), and NAGA (*p=0.032)) between R1 and R2 sorted using FACS and analyzed using qPCR. (F) Differential protein expression analysis (NAGA, LYZ1, and NLRP3) between R1 and R2 sorted using FACS. The immunoblot signals were quantified with ImageJ. The relative immunoblot signal intensities of NAGA (*p=0.011), LYZ1 (*p=0.022), and NLRP3 (**p=0.009) compared with that of ß-actin were recorded with bar chart. Both qPCR and immunoblot data were analyzed using the student t test.

