The characterization of an agranulocyte-specific marker (CgCD9) in the Pacific oyster Crassostrea gigas

The agranulocytes in the Pacific oyster Crassostrea gigas are a group of haemocytes that are significantly different from semi-granulocytes and granulocytes on the morphology. Agranulocytes are the smallest haemocytes characterized by a spherical shape, the largest ratio of nucleus to cytoplasm, and no granules in the cytoplasm. The lack of unique cell surface markers impedes the isolation of agranulocytes from total haemocytes. Previous transcriptome sequencing analysis of three subpopulations of haemocytes revealed that a homologue of CD9 (designed as CgCD9) was highly expressed in agranulocytes of oyster C. gigas (data not shown). In the present study, CgCD9 was identified to share a similarity of 60% with other vertebrates CD9s, and it harbored a typical four transmembrane domain and a conserved Cys-Cys-Gly (CCG) motif. The mRNA transcript of CgCD9 was found to be highly expressed in agranulocytes, which was 6.63-fold (p < 0.05) and 3.68-fold (p < 0.05) of that in granulocytes and semi-granulocytes, respectively. A specific monoclonal antibody of CgCD9 (named 3D8) was successfully prepared by traditional hybridoma technology, and a single positive band at 25.2 kDa was detected in the haemocyte proteins by Western Blotting, indicating that this monoclonal antibody exhibited high specificity and sensitivity to CgCD9 protein. The ELISA positive value of 3D8 monoclonal antibody to recognize agranulocytes, semi-granulocytes and granulocytes was 17.35, 4.48 and 1.55, respectively, indicating that monoclonal antibody was specific to agranulocytes. Immunocytochemistry assay revealed that CgCD9 was specifically distributed on the membrane of agranulocytes. Using immunomagnetic beads coated with 3D8 monoclonal antibody, CgCD9+cells with a purity of 94.53 ± 5.60% were successfully isolated with a smaller diameter, a larger N:C ratio and no granules in cytoplasm, and could be primary culture in the modified L-15 medium in vitro. Collectively, these results suggested that CgCD9 was a specific cell surface marker for agranulocytes, which offered a tool for high-purity capture of agranulocytes from total haemocytes in C. gigas.