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A new protocol for the simultaneous flow cytometric analysis of cytotoxicity and immunotoxicity on zebra mussel (Dreissena polymorpha) hemocytes.
Fish & Shellfish Immunology ( IF 4.7 ) Pub Date : 2019-12-30 , DOI: 10.1016/j.fsi.2019.12.092
Iris Barjhoux 1 , Damien Rioult 2 , Alain Geffard 1 , Melissa Palos Ladeiro 1
Affiliation  

Immunotoxicity analysis receives a strong interest in environmental a priori and a posteriori risk assessment procedures considering the direct involvement of the immune system in the health status of organisms, populations and thus ecosystems. The freshwater mussel Dreissena polymorpha is an invasive species widely used in ecotoxicology studies and biomonitoring surveys to evaluate the impacts of contaminants on aquatic fauna. Bivalve hemocytes are the immunocompetent cells circulating in the open circulatory system of the organism. However, there is nowadays no consensus on a protocol to evaluate the immunocompetent state of this particular cell type using flow cytometry. Wild species such as D. polymorpha present several technical barriers complicating their analyze including (i) the quality and the purity of the hemolymph sample, (ii) the controversial characterization of hemocyte subpopulations and their diversity, (iii) the quantity of biological material, and (iv) the high inter-individual variability of hemocyte responses. The present work proposes several technical and analytical improvements to control the above-mentioned issues. The inclusion of sedimentation and cell detachment steps in the pre-analytical phase of the protocol substantially ameliorate the quality of the hemolymph sample as well as the accuracy of the cytometric measurements, by selecting the analyzed cells on their adhesion ability and by increasing the concentration of the analyzed events. The development of an effective triple-labeling procedure including the cellular probe Hoechst® 33342, the membrane impermeant dye propidium iodide and yellow-green fluorescent microspheres allowed the simultaneous analysis of cytotoxicity and phagocytosis activity in hemocytes. It also significantly enhanced the accuracy of hemocyte endpoint measurements by eliminating non-target events from the analysis and allowing relevant gating strategies. Finally, the use of pooled samples of hemolymph noticeably reduced inter-sample variability while providing more plasticity in the experimental design and improving the discriminating potency between treatments. The developed protocol is suitable for ex vivo exposure of hemocyte in a chemical/environmental toxicity assessment as well as for in vivo exposure in the laboratory or in situ biomonitoring surveys with few adaptations.

中文翻译:

同时对斑马贻贝(Dreissena polymorpha)血细胞的细胞毒性和免疫毒性进行流式细胞术分析的新协议。

考虑到免疫系统直接参与生物体,种群以及生态系统的健康状况,免疫毒性分析在先验环境和后验风险评估程序中引起了浓厚的兴趣。淡水贻贝Dreissena polymorpha是一种入侵物种,广泛用于生态毒理学研究和生物监测调查,以评估污染物对水生动物的影响。双壳类血细胞是在生物体的开放循环系统中循环的免疫活性细胞。然而,如今在使用流式细胞仪评估该特定细胞类型的免疫能力状态的方案上尚无共识。野生种(如多形衣藻)存在多种技术障碍,使它们的分析复杂化,其中包括(i)血淋巴样品的质量和纯度,(ii)血细胞亚群及其多样性的争议性特征,(iii)生物材料的数量,以及(iv)血细胞反应的个体间差异很大。本工作提出了一些技术和分析方面的改进,以控制上述问题。通过选择分析细胞的黏附能力并增加其浓度,该方案的分析前阶段包括沉淀和细胞分离步骤,可大大改善血淋巴样品的质量以及细胞计数法的准确性。分析的事件。开发出有效的三标记程序,包括细胞探针Hoechst®33342,膜不渗透染料碘化丙锭和黄绿色荧光微球可以同时分析血细胞的细胞毒性和吞噬活性。通过从分析中消除非目标事件并允许相关的门控策略,它还显着提高了血细胞终点测量的准确性。最后,使用合并的血淋巴样本可显着降低样本间的变异性,同时在实验设计中提供更多的可塑性,并提高治疗之间的区分力。该开发的协议适用于化学/环境毒性评估中的血细胞离体暴露,以及适应性极低的实验室或原位生物监测调查中的体内暴露。通过从分析中消除非目标事件并允许相关的门控策略,它还显着提高了血细胞终点测量的准确性。最后,使用合并的血淋巴样品可显着降低样品间的变异性,同时在实验设计中提供更多的可塑性,并提高治疗之间的区分力。该开发的协议适用于化学/环境毒性评估中的血细胞离体暴露,以及适应性极低的实验室或原位生物监测调查中的体内暴露。通过从分析中消除非目标事件并允许相关的门控策略,它还显着提高了血细胞终点测量的准确性。最后,使用合并的血淋巴样本可显着降低样本间的变异性,同时在实验设计中提供更多的可塑性,并提高治疗之间的区分力。该开发的协议适用于化学/环境毒性评估中的血细胞离体暴露,以及适应性极低的实验室或原位生物监测调查中的体内暴露。使用合并的淋巴液样本可显着降低样本间的变异性,同时在实验设计中提供更大的可塑性,并提高治疗之间的区分力。该开发的协议适用于化学/环境毒性评估中的血细胞离体暴露,以及适应性极低的实验室或原位生物监测调查中的体内暴露。使用合并的淋巴液样本可显着降低样本间的变异性,同时在实验设计中提供更大的可塑性,并提高治疗之间的区分力。该开发的协议适用于化学/环境毒性评估中的血细胞离体暴露,以及适应性极低的实验室或原位生物监测调查中的体内暴露。
更新日期:2019-12-30
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