Díaz-Bello Beatriz, Arawi Dalia El, Torro Rémy, Chames Patrick, Sengupta Kheya, Limozin Laurent
Aix-Marseille University, CNRS, INSERM, LAI, Turing Center for Living Systems, Marseille, France.
Aix-Marseille University, CNRS, CINAM, Turing Center for Living Systems, Marseille, France.
Bio Protoc. 2025 Sep 5;15(17):e5427. doi: 10.21769/BioProtoc.5427.
Cell-surface and cell-cell interaction assays are fundamental for studying receptor-ligand interactions and characterizing cellular responses and functions. They play a critical role in diagnostics and in modulating immune system activity for therapeutic applications, notably in cancer immunotherapy. By providing time-lapsed and cell-level direct observation of the sample, optical microscopy offers strong advantages compared to current go-to techniques, which are typically either ensemble methods (e.g., measuring cell populations) or indirect readouts (e.g., impedance for adherent cells). This protocol describes two complementary microscopy-based assays: (1) a cell-surface ligand binding assay to quantify dynamic interactions between human primary Natural Killer (NK) cells and a cancer-mimicking surface, and (2) a cell-cell interaction assay to evaluate antibody-dependent cell cytotoxicity (ADCC) mediated by NK cells targeting tumor cells. Additionally, the protocol uses Celldetective, a new open graphical user interface for quantitative analysis of cell interaction dynamics from 2D time-lapse microscopy datasets. Although applied here to primary immune cells, these methods are adaptable to various cell types, including other immune cells, fibroblasts, and cancer cells. This approach enables direct observation and quantification of cellular morphology, motility, cell-cell interactions, and dynamic behaviors at single-cell resolution over time, facilitating detailed analysis of mechanisms such as cell death, migration, and immune synapse formation. Key features • End-to-end protocol for antibody evaluation by optical microscopy on living cells using simple reagents, followed by full open-source software image analysis and data rendering • Quantitative analysis of cell-surface interactions using label-free imaging to study the dynamic spreading of NK cells on antibody-coated surfaces under different antibody concentrations. • High-resolution evaluation of antibody-dependent cell cytotoxicity in NK-cancer cells co-culture using fluorescence imaging, deep learning-based death detection, and synchronized single-cell measurements.
细胞表面和细胞间相互作用分析对于研究受体 - 配体相互作用以及表征细胞反应和功能至关重要。它们在诊断以及调节免疫系统活性以用于治疗应用（尤其是癌症免疫治疗）中发挥着关键作用。通过对样本进行延时和细胞水平的直接观察，光学显微镜与当前常用技术相比具有显著优势，当前常用技术通常要么是整体方法（例如测量细胞群体），要么是间接读数（例如贴壁细胞的阻抗）。本方案描述了两种基于显微镜的互补分析方法：（1）一种细胞表面配体结合分析，用于量化人原代自然杀伤（NK）细胞与模拟癌症表面之间的动态相互作用；（2）一种细胞间相互作用分析，用于评估由靶向肿瘤细胞的NK细胞介导的抗体依赖性细胞毒性（ADCC）。此外，该方案使用了Celldetective，这是一个新的开放图形用户界面，用于从二维延时显微镜数据集中定量分析细胞相互作用动力学。尽管此处应用于原代免疫细胞，但这些方法适用于各种细胞类型，包括其他免疫细胞、成纤维细胞和癌细胞。这种方法能够在单细胞分辨率下随时间直接观察和量化细胞形态、运动性、细胞间相互作用和动态行为，便于详细分析细胞死亡、迁移和免疫突触形成等机制。关键特性 • 使用简单试剂通过光学显微镜对活细胞进行抗体评估的端到端方案，随后进行完全开源的软件图像分析和数据呈现 • 使用无标记成像对细胞表面相互作用进行定量分析，以研究不同抗体浓度下NK细胞在抗体包被表面上的动态铺展。 • 使用荧光成像、基于深度学习的死亡检测和同步单细胞测量，对NK - 癌细胞共培养中的抗体依赖性细胞毒性进行高分辨率评估。
