光路系统的优化配置在多色流式细胞术实验方案设计中的应用

Optimal configuration of optical path system for designing multicolor flow cytometry

目的:基于流式细胞仪光路系统的硬件配置情况,探讨多色流式细胞术实验方案的优化设计。方法:选用6~8周B6小鼠的骨髓细胞作为实验材料,在已有流式抗体的基础上,通过变更BD AriaⅢ流式细胞仪中的滤光片,分别检测各荧光通道信号的相对变异系数%rCV和荧光强度中位值(median of fluorescence intensity,MFI)以及小鼠骨髓中的造血干细胞(hematopoietic stem cell,HSC)、造血祖细胞(hematopoietic progenitor cell,HPC)和多能祖细胞(multipotent progenitor cell,MPP)的活性氧(reactive oxygen species,ROS)水平。结果:通过优化光路系统配置,滤光片变更前后各荧光通道的%rCV无明显差异;HSC、HPC、MPP等细胞亚群在PerCP-Cy5.5通道的MFI(ROS水平)均增大;各亚群PerCP-Cy5.5通道的荧光信号(ROS水平)整体右移,实现了多参数流式分析。结论:在多色流式细胞术实验方案设计中,如相关荧光素抗体搭配受限,可通过灵活搭配使用流式细胞仪中的滤光片,合理优化光路系统的硬件配置,达到完成多色流式细胞术检测的目的。

Objective To explore the optimal design of multicolor flow cytometry experimental protocols based on the hardware configuration of the flow cytometry optical path system.Methods Bone marrow cells from 6-8 week old B6 mice were used as the experimental material.The relative coefficient of variation(%rCV)and median of fluorescence intensity(MFI)of each fluorescence channel signal and the reactive oxygen species(ROS)of hematopoietic stem cells(HSC),hematopoietic progenitor cells(HPC)and multipotent progenitor cells(MPP)in the bone marrow were measured by changing the filter in the BD AriaⅢflow cytometer based on the existing flow antibodies.Results Configuration optimization of the optical system made the%rCV of each fluorescence channel had no significant differences before and after changing the filter;all the MFIs(ROS level)of HSC,HPC and MPP cell subpopulations in the PerCP-Cy5.5 channel increased;the fluorescence signal(ROS level)of each subpopulation PerCP-Cy5.5 channel was shifted to the right as a whole,realizing the multi-parameter flow analysis.Conclusion During the design of multicolor flow cytometry protocols when the matching of fluorescein antibodies is limited,the filters in the flow cytometer can be used flexibly to optimize the hardware configuration of the optical path system to fulfill multicolor flow cytometry assays.