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目的:改良常规免疫磁性活化细胞分选(MACS)的分离纯化方法,提高分选后细胞的纯度并确保细胞仍具有良好活性及功能。方法:以干细胞抗原-1(Sca-1)标记的Sca-1+造血干/祖细胞(Sca-1+HSC/HPC)为例,分别用改良后和常规MACS法分选出小鼠骨髓细胞Sca-1+细胞,流式细胞术检测两种分选方法获得Sca-1+细胞纯度;计算阳性细胞回收率;台酚蓝染色检测分选细胞存活百分率;CCK-8检测细胞增殖,混合造血祖细胞(CFU-Mix)体外培养评价分选Sca-1+细胞分化能力。结果:改良MACS法分选获得的Sca-1+细胞纯度达93%以上(常规MACS法仅为87%),阳性细胞的回收率可达73%(常规法仅为62.3%);细胞存活率、细胞增殖和CFU-Mix集落形成能力两种分选方法无明显差别。结论:改良法能明显提高细胞分选纯度及回收率,细胞活性和功能保持良好,值得各种细胞免疫磁性分选参考采用。
OBJECTIVE: To improve the separation and purification of conventional immunomagnetic activated cell sorting (MACS), improve the purity of cells after sorting and ensure that the cells still have good activity and function. Methods: Sca-1 + HSC / HPC labeled with Sca-1 was used as an example. Mature bone marrow cells Sca-1 + cells, flow cytometry detection of two kinds of sorting methods to obtain Sca-1 + cell purity; calculate the recovery of positive cells; Taiwan Phenol blue staining sorting cell survival percentage; CCK-8 detection of cell proliferation, mixed hematopoietic Progenitor cells (CFU-Mix) were cultured in vitro to evaluate the ability of Sca-1 + cells to differentiate. Results: The purity of Sca-1 + cells obtained by modified MACS method was more than 93% (only 87% by conventional MACS method), and the recovery rate of positive cells was 73% (conventional method was only 62.3%). The cell survival rate There was no significant difference between the two methods of cell proliferation and CFU-Mix colony forming ability. Conclusion: The modified method can significantly improve cell sorting purity and recovery, cell activity and function remain well, it is worth a variety of cellular immune magnetic separation reference.