Rieger Aja M, Nelson Kimberly L, Konowalchuk Jeffrey D, Barreda Daniel R
Department of Biological Sciences, University of Alberta.
J Vis Exp. 2011 Apr 24(50):2597. doi: 10.3791/2597.
Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability. The Annexin V/PI protocol is a commonly used approach for studying apoptotic cells. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).
自基于流式细胞术的方法问世以来，细胞凋亡研究受到了重大影响。碘化丙啶（PI）广泛与膜联蛋白V联合使用，通过质膜完整性和通透性的差异来确定细胞是存活、凋亡还是坏死。膜联蛋白V/PI方案是研究凋亡细胞的常用方法。PI比其他核染色剂使用得更频繁，因为它经济、稳定，并且基于其在活细胞中排除染料的能力，是细胞活力的良好指标。PI进入细胞的能力取决于膜的通透性；由于存在完整的质膜，PI不会对活细胞或早期凋亡细胞进行染色。在晚期凋亡和坏死细胞中，质膜和核膜的完整性降低，使得PI能够穿过膜，插入核酸并发出红色荧光。不幸的是，我们发现传统的膜联蛋白V/PI方案会导致大量假阳性事件（高达40%），这些事件与细胞质区域内RNA的PI染色有关。广泛动物模型中的原代细胞和细胞系都会受到影响，大细胞（核：质比<0.5）的发生率最高。在此，我们展示了一种改进的膜联蛋白V/PI方法，与传统方法相比，该方法在评估细胞死亡方面有显著改进。该方案利用细胞固定过程中细胞通透性的变化，促进RNase A在染色后进入细胞。RNase A的时间和浓度都已优化，以去除细胞质RNA。结果是与传统的膜联蛋白V/PI方案相比有显著改进（细胞质PI染色的事件<5%）。
