溃疡性结肠炎 (UC) 和克罗恩病 (CD) 等炎症性肠病 (IBD) 患者的肠上皮细胞 (IEC) 死亡增加。这可能导致肠道屏障功能缺陷、炎症加剧和疾病免疫发病机制。细胞因子和死亡受体配体是 IEC 死亡增加的部分原因。 IBD 相关细胞因子，例如 TNF-α 和 IFN-γ，无论单独还是联合使用，都对 IEC 具有细胞毒性。该协议描述了一种简单实用的测定方法，使用荧光细胞死亡染料（SYTOX 绿色核酸染色）、活体荧光显微镜和开源图像分析软件来量化 CD 患者来源的结肠类器官中细胞因子诱导的细胞毒性。我们还演示了如何使用 Bliss 独立数学模型来计算基于类器官细胞毒性的扰动相互作用系数 (CPI)。 CPI 可用于确定细胞因子组合或其他类型的扰动因素之间的相互作用是拮抗的、相加的还是协同的。该协议可用于使用患者来源的结肠类器官来研究细胞因子和其他扰动物的细胞毒性活性。

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-α and IFN-γ, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.