Institute for Health Innovation and Technology, National University of Singapore, Singapore, Singapore.
Critical Analytics for Manufacturing of Personalized-Medicine Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore, Singapore.
Nat Biomed Eng. 2024 Mar;8(3):248-262. doi: 10.1038/s41551-023-01089-z. Epub 2023 Aug 31.
Assays for assessing cell-mediated cytotoxicity are largely target-cell-centric and cannot identify and isolate subpopulations of cytotoxic effector cells. Here we describe an assay compatible with flow cytometry for the accurate identification and sorting of functional killer-cell subpopulations in co-cultures. The assay, which we named PAINTKiller (for 'proximity affinity intracellular transfer identification of killer cells'), relies on the detection of an intracellular fluorescent protein 'painted' by a lysed cell on the surface of the lysing cytotoxic cell (specifically, on cell lysis the intracellular fluorescein derivative carboxyfluorescein succinimidyl ester is captured on the surface of the natural killer cell by an antibody for anti-fluorescein isothiocyanate linked to an antibody for the pan-leucocyte surface receptor CD45). The assay can be integrated with single-cell RNA sequencing for the analysis of molecular pathways associated with cell cytotoxicity and may be used to uncover correlates of functional immune responses.
用于评估细胞介导的细胞毒性的测定法主要以靶细胞为中心，无法识别和分离细胞毒性效应细胞的亚群。在这里，我们描述了一种与流式细胞术兼容的测定法，用于准确识别和分选共培养物中功能性杀伤细胞亚群。该测定法称为 PAINTKiller（表示“通过亲和性内转移识别杀伤细胞”），依赖于对细胞毒性细胞表面上裂解细胞“绘制”的细胞内荧光蛋白的检测（具体而言，在细胞裂解时，细胞内荧光素衍生物羧基荧光素琥珀酰亚胺酯通过与抗荧光素异硫氰酸酯连接的抗体被捕获到自然杀伤细胞表面上，该抗体连接到针对白细胞表面受体 CD45 的抗体上）。该测定法可以与单细胞 RNA 测序集成，用于分析与细胞毒性相关的分子途径，并且可以用于发现与功能性免疫反应相关的关联。
