Pierce Mackenzie, Huang Yongyang, Lin Allen, Franco Nitta Carolina, Kuksin Dmitry, Lin Bo, Chan Leo Li-Ying
Department of Advanced Technology R&D, Revvity Health Sciences, Inc., 360 Merrimack St., Suite 200, Lawrence, MA, 01843, USA.
J Fluoresc. 2025 Feb;35(2):1111-1123. doi: 10.1007/s10895-024-03590-3. Epub 2024 Jan 31.
Apoptosis is the programmed cell death pathway that is critical for maintaining homeostasis, in which cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare different cancer therapeutics (i.e., small molecules, antibody drugs, cell therapies) that can initiate the process of apoptosis, enabling the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V, Caspase-3, and Propidium Iodide (PI) using the Cellaca® PLX Image Cytometer (Revvity Health Sciences, Inc., Lawrence, MA). First, apoptosis was induced in Jurkat and K562 cell lines with staurosporine over the course of 24 h, where apoptosis and necrosis were assessed at 0, 1, 1.5, 2, 4, 20, and 24 h timepoints. Samples were stained with Hoechst 33342 (total dye), Annexin V-APC (early-stage apoptosis), Caspase-3 488 (late-stage apoptosis), and PI (necrosis) at each timepoint and evaluated using image cytometry. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early- to late-stage apoptosis, and ultimately necrosis. A clear trend was observed analyzing apoptotic and necrotic populations during the first 1.5 h, showing differences of up to ~15% in single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis between apoptotic populations only, Annexin V+ only populations were higher than Caspase-3+ only populations by up to ~20% between 0 and 1.5 h. Conversely, K562 cells did not exhibit a notable change in apoptotic and necrotic populations due to low sensitivity to staurosporine. The proposed image cytometric detection method may provide an effective and efficient tool for rapid and reliable simultaneous detection of early- late-stage apoptosis, and necrosis. Therefore, allowing researchers to better characterize and screen potential cancer therapeutic drug candidates for their treatment efficacy in a higher throughput manner.
细胞凋亡是一种程序性细胞死亡途径，对维持体内平衡至关重要，而癌细胞可以逃避这种途径以确保存活。对于药物研发而言，表征和比较不同的癌症治疗方法（即小分子、抗体药物、细胞疗法）以启动细胞凋亡过程非常重要，这有助于识别潜在的治疗候选物。在这项工作中，我们开发并展示了一种多重检测方法，使用Cellaca® PLX图像细胞仪（Revvity健康科学公司，马萨诸塞州劳伦斯），通过膜联蛋白V、半胱天冬酶-3和碘化丙啶（PI）来监测细胞凋亡和坏死。首先，用星形孢菌素在24小时内诱导Jurkat和K562细胞系发生凋亡，并在0、1、1.5、2、4、20和24小时时间点评估细胞凋亡和坏死情况。在每个时间点，样本用Hoechst 33342（总染料）、膜联蛋白V-APC（早期凋亡）、半胱天冬酶-3 488（晚期凋亡）和PI（坏死）进行染色，并使用图像细胞术进行评估。结果表明，沿着从早期凋亡到晚期凋亡，最终到坏死的途径成功检测到了凋亡因子和级联反应。在最初的1.5小时内分析凋亡和坏死细胞群体时观察到了明显的趋势，在处理过的Jurkat细胞中，单一膜联蛋白V+和半胱天冬酶-3+细胞群体的差异高达约15%，然而，直到20小时才观察到膜联蛋白V+PI+和半胱天冬酶-3+PI+双阳性凋亡/坏死细胞的显著增加。仅在凋亡细胞群体之间进行进一步分析时，在0到1.5小时之间，仅膜联蛋白V+细胞群体比仅半胱天冬酶-3+细胞群体高出约20%。相反，由于对星形孢菌素敏感性较低，K562细胞在凋亡和坏死细胞群体中未表现出明显变化。所提出的图像细胞术检测方法可能为快速、可靠地同时检测早期到晚期凋亡及坏死提供一种有效且高效的工具。因此，使研究人员能够以更高通量的方式更好地表征和筛选潜在的癌症治疗药物候选物的治疗效果。
