| In this study, homogenization, centrifugation, microscopy, and marker enzyme assays were used to study the separation and identification techniques for subcellular fractions of the digestive gland tissues of Chlamys farreri. The results showed that after Hoechst 33258 staining, a large number of intact cells, which were round or elliptical, with complete cell membrane and high fluorescence intensity, were observed in the 2 min homogenization group. In the 3, 4, and 5 min homogenization group, the number of intact cells gradually decreased, and many cell fragments with small morphology, weak fluorescence, and blurred outline appeared. Additionally, the blood cell counting results were consistent with the Hoechst staining results. With the increase in homogenization time (2~5 min), the broken cell rate was increased under an optical microscope and when the 2 min homogenization group was used as the control group, the broken cell rate reached 94.24% in the 5 min homogenization group. There were more fluorescent particles in C2 with appropriate size, and clear structure, and fewer fluorescent particles were observed in other separated fractions, when fractions (S2, C2, C4, S5, and C5) of the digestive gland tissues of C. farreri were stained with Hoechst 33258. Therefore, the nucleus mainly presented in the C2 fraction. At the same time, the marker enzyme activity of the cell membrane (5ʹ-nucleotidase), mitochondria (succinate dehydrogenase), cytoplasmic (lactate dehydrogenase), and microsomes (glucose-6-phosphatase) accounted for a high proportion (63.90%, 64.89%, 77.82%, and 67.55%) in the S2, C4, S5, and C5 fractions, respectively; however, there were also a small amount found in other subcellular fractions. As a result, the separated fractions of S2, C2, C4, S5, and C5 were cell membrane, nucleus, mitochondria, cytoplasm, and microsomes, respectively, and the technical methods for separation and identification of subcellular fractions of the digestive gland tissues of C. farreri were successfully determined. |
