Application of imaging flow cytometry in studies of the classification and phagocytic function of Eriocheir sinensis blood cell

In order to make more accurate classification of crustacean blood cells and understand their phagocytic function more clearly, an imaging flow cytometry-based automatic method for Eriocheir sinensis blood cell classification was established, and this new method was further compared with microscopic observation for evaluating their abilities in classifying blood cells through characteristic differences of cytoplasmic granular structure in cells. On this basis, 7.5×10⁸ of Thermo Fisher carboxylate-modified yellow-green fluorescent microspheres with 1 μm diameter were injected through the heart of E. sinensis of (10±3) g weight; proportions of cells containing engulfed microspheres in hemolymph were measured by imaging flow cytometry at 2, 4, 6 and 24 h after injection to analyze the phagocytic efficiency and foreign-body-removing ability of blood cells. The results showed that both microscopic observation and imaging flow cytometry-based classification methods could distinguish 4 blood cell populations from hemolymph of E. sinensis, but the population proportions from both methods were different. The possible reasons might be that populations in microscopic observing classification were divided based on the observable granular structures in each cell, but populations got from imaging flow cytometry method were distinguished by all measurable granular structures in each cell. However, due to lack of quantified specific classification standard, the identification results from microscopic observing were fluctuant and what’s more, this work was also time-consuming and laborious compared with automatic methods. On the contrary, imaging flow cytometry measured all detectable intracellular particulate matters. With updated detection module, redesigned flowing system, and, what’s more, innovative integration of photographic function for each detecting channel, imaging flow cytometry could offer a high-throughput detection, and the data were more objective and reliable than those of manual distinguishing and counting. According to the flow cytometry analyzing, non-granular hemocyte, small-granular hemocyte, intermediate-granular hemocyte, large-granular hemocyte accounted for 40.62%±2.65 %, 36.68%±6.84 %, 7.80%±1.16 % and 16.51%±5.60 % separately of total blood cells from E. sinensis. Lack of transition dots between populations means there are no cell type transformations among 4 cell populations. Phagocytic function of E. sinensis blood cells was analyzed by both of above methods. According to microscopic observing results, all 4 kinds of blood cells could engulf fluorescent microspheres. The less the number of granules in the blood cells, the stronger the phagocytic capability of the blood cells. So, the non-granular hemocytes were the main phagocytic population. From the results of imaging flow cytometry method, proportion of blood cells containing microsphere went up in 2 hours after injection, and quickly reached a peak of 5.69%±0.44% at 4 hours, then followed by a rapid decline during 4–6 hours after injection, which means that E. sinensis blood cells were effective in removing foreign matter from the hemolymph. As it is known that fluorescent microspheres are difficult to be degraded by enzymes in cells, the declining proportion of cells containing microspheres is likely to be caused by such mechanisms as agglutination and immobilization of cycling cells, which help cells leave the E. sinensis’s circulatory system. Imaging flow cytometry is suitable for the classification researches and functional studies of E. sinensis blood cells, it would be more effective when combined with microscopic observation. The blood cells of E. sinensis have strong phagocytic ability. Probably through cell agglutination, E. sinensis could quickly remove invaded foreign matter, which, usually, is difficult to be degraded. These results provide new and important references for related researches, and will facilitate to understand the functions of blood cells in crustaceans more clearly.