Genomics and Applied Biology 2018, Vol.9, No.9, 56-61
60
solution to prepare Percoll working fluid. The Percoll working fluid was formulated into 34% and 51%
concentration. We took a 15 mL centrifuge tube with 51% lower layer Percoll concentration and 34% upper layer
Percoll concentration. Upper layer Percoll was added gently and it was better to have clear liquid level without
fluctuation. Then the cell suspension prepared in 1.2.1 was added to the upper layer Percoll, it was better to have
clear liquid level without fluctuation. The volume of the two-layer Percoll was 2 mL, and the volume of the added
cell suspension was 4 mL. The Percoll was centrifuged and the time was set to 30 minutes. The rotation speed was
500 g with the temperature of 4°C, and the speed of rise and fall was slow. The target cell layer was aspirated and
placed in a new centrifuge tube. Then, we added PBS 2 mL and the Percoll was centrifuged for 4 minutes with the
rotation speed of 300 g. The rising and falling speed was set to normal, and the supernatant was discarded. 2 mL
PBS suspension and primary antibody was added to incubate on ice for 0.5 h. 2 mL PBS was added to wash out
the uncombined primary antibody, and the Percoll was centrifuged for 4 minutes, setting the rotation speed to 300 g.
The rising and falling speed was set to normal, and the supernatant was discarded. Next, we added the second
antibody and incubated on ice without light for 0.5 h. 2 mL PBS was added, and the Percoll was centrifuged for
6 minutes with rotating speed of 300 g. The rising and falling speed was normal, and the supernatant was
discarded. PBS was added to make lymphocyte suspension. Through flow cytometry analysis, we knew the
distribution of lymphocyte groups and found that NCC particle size was smaller than neutrophils and monocytes.
The target cells were found in the flow cytometry (Figure 2A; Figure 2C).
Authors’ contributions
ZQ was responsible for the separation of NCC from
Oreochromis niloticus
, immunohistochemical staining and paper writing.
Professor JJC was the project manager and guided students to study. YM was responsible for the immunohistochemistry of four
tissues of
Oreochromis niloticus
. HY was responsible for providing antibody and experimental guidance. Associate professor CJ was
responsible for designing experiments. All authors read and approved the final manuscript.
Acknowledgments
This study was co-funded by the National Natural Science Foundation of China (31572651), Guangdong Science and Technology
Project (2015A020209181) and Guangdong Ocean University Doctoral Dissertation Cultivation Project (201602).
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