Aquaculture Biotechnology Research Group, School of Science, Faculty of Health and Environmental Sciences, Auckland University of Technology, Private Bag 92006, Auckland, 1142, Auckland, New Zealand.
AUT-Roche Diagnostics Laboratory, School of Science, Auckland University of Technology, Private Bag 92006, Auckland, 1142, Auckland, New Zealand.
Fish Shellfish Immunol. 2018 Jul;78:131-139. doi: 10.1016/j.fsi.2018.04.038. Epub 2018 Apr 21.
The ability to successfully prepare and preserve haemocyte cells for microscopy and flow cytometry is critical for the investigation of animal immune systems. In this study, we observed the total cell count, in vitro viability and stability of New Zealand black-footed abalone (Haliotis iris) haemocytes with different antiaggregants and handling protocols. Haemocyte stability was evaluated by direct observation of haemocytes under the microscope and calculating the aggregation index. Haemocyte counts and viability were measured via flow cytometry and tested for the effect of different antiaggregants (Alsever's solution at three concentrations, and specialised blood collection tubes containing lithium heparin and KEDTA) at different temperatures and storage times. Results showed that Alsever's solution is an effective antiaggregant at haemolymph:antiaggregant dilution ratios of 1:1, 1:2 and 1:3. Lithium heparin was ineffective as an antiaggregant, whereas KEDTA was similarly as effective as Alsever's solution. The influence of different mixing techniques (vortex, pipetting and flipping) were subsequently tested using the KEDTA Microtainer tubes, revealing that proper mixing should be performed immediately. High cell viability can be achieved by mixing samples by either 10 s of vortexing (1000 rpm), 10 times pipetting or 20 times flipping. The in vitro storage of abalone haemocytes in AS and KEDTA as antiaggregants at ambient room temperature was highly effective for up to 24 h (75-85% viability; 0.05-0.15 aggregation index) and is recommended for haemocyte studies in H. iris. Utilization of KEDTA Microtainer tubes were advantageous since they are more cost effective compared to Alsever's solution, and samples can be prepared more efficiently.
成功地准备和保存血细胞用于显微镜和流式细胞术分析对于研究动物免疫系统至关重要。在这项研究中，我们观察了不同的抗聚集剂和处理方案对新西兰黑足鲍（Haliotis iris）血细胞的总细胞计数、体外活力和稳定性的影响。通过在显微镜下直接观察血细胞并计算聚集指数来评估血细胞的稳定性。通过流式细胞术测量血细胞计数和活力，并测试不同抗聚集剂（三种浓度的 Alsever 溶液，以及含有肝素锂和 KEDTA 的专用血液采集管）在不同温度和储存时间下的效果。结果表明，Alsever 溶液在血淋巴：抗聚集剂稀释比例为 1:1、1:2 和 1:3 时是一种有效的抗聚集剂。肝素锂作为抗聚集剂无效，而 KEDTA 的效果与 Alsever 溶液相似。随后使用 KEDTA Microtainer 管测试了不同混合技术（涡旋、移液和翻转）的影响，结果表明应立即进行适当的混合。通过涡旋（1000rpm）10 秒、10 次移液或 20 次翻转的方式混合样品，可以获得高的细胞活力。在室温下，AS 和 KEDTA 作为抗聚集剂在体外储存鲍血细胞长达 24 小时（活力 75-85%；聚集指数 0.05-0.15）的效果非常好，推荐用于 H. iris 的血细胞研究。与 Alsever 溶液相比，KEDTA Microtainer 管更具成本效益，且样品制备效率更高，因此具有优势。
