Siniyeh Ahmed Abu, Alshaer Walhan, Elzogheir Nirmeen, Al-Holi Majed, Alqudah Dana A, Abuarqoub Duaa, Kwiatek Joanna M
Department of Medical Laboratory Sciences, School of Science, The University of Jordan, Amman, Jordan.
Cell Therapy Center, The University of Jordan, Amman, Jordan.
Biochem Biophys Rep. 2025 Aug 30;44:102225. doi: 10.1016/j.bbrep.2025.102225. eCollection 2025 Dec.
This study evaluates the effectiveness of fluorescence microscopy using Di-4-ANEPPDHQ in differentiating macrophage phenotypes (M0, M1, and M2) compared to RT-qPCR and flow cytometry. Using THP-1 monocyte-derived macrophages, we assessed cytokine expression (IL-1β, IL-6, IL-10) via RT-qPCR, surface markers (CD86, CD64, CD206) through flow cytometry, and membrane properties with Di-4-ANEPPDHQ fluorescence. RT-qPCR showed significant differences in cytokine expression: M1 macrophages had elevated IL-1β (p < 0.0001) and IL-6 (p < 0.0001), while M2 macrophages exhibited higher IL-10 levels (p = 0.0030). Flow cytometry revealed distinct surface marker profiles, with M1 expressing high CD64 and M2 showing increased CD206. Di-4-ANEPPDHQ fluorescence indicated membrane order differences: M1 macrophages were depolarized (red shift), while M2 macrophages were hyperpolarized (blue shift). Statistical analysis confirmed high sensitivity and specificity for RT-qPCR and flow cytometry, while Di-4-ANEPPDHQ fluorescence technique provides real-time observations of changes in macrophage membrane behavior, enhancing understanding of their dynamic properties under various conditions. These findings highlight the value of integrating these methods for comprehensive macrophage phenotype characterization, which can aid in understanding macrophage polarization in immune responses and disease contexts.
本研究评估了与逆转录定量聚合酶链反应（RT-qPCR）和流式细胞术相比，使用Di-4-ANEPPDHQ的荧光显微镜在区分巨噬细胞表型（M0、M1和M2）方面的有效性。我们使用THP-1单核细胞衍生的巨噬细胞，通过RT-qPCR评估细胞因子表达（白细胞介素-1β、白细胞介素-6、白细胞介素-10），通过流式细胞术检测表面标志物（CD86、CD64、CD206），并用Di-4-ANEPPDHQ荧光检测膜特性。RT-qPCR显示细胞因子表达存在显著差异：M1巨噬细胞的白细胞介素-1β（p < 0.0001）和白细胞介素-6（p < 0.0001）升高，而M2巨噬细胞的白细胞介素-10水平更高（p = 0.0030）。流式细胞术揭示了不同的表面标志物谱，M1表达高CD64，M2显示CD206增加。Di-4-ANEPPDHQ荧光表明膜有序性存在差异：M1巨噬细胞去极化（红移），而M2巨噬细胞超极化（蓝移）。统计分析证实了RT-qPCR和流式细胞术具有高灵敏度和特异性，而Di-4-ANEPPDHQ荧光技术可实时观察巨噬细胞膜行为的变化，增强对其在各种条件下动态特性的理解。这些发现突出了整合这些方法用于全面表征巨噬细胞表型的价值，这有助于理解免疫反应和疾病背景下的巨噬细胞极化。
