Hoechst 33258/PI细胞凋亡染色试剂盒是一种采用Hoechst 33258和碘化丙啶(Propidium Iodide，PI)双荧光染色方法以区分凋亡细胞和坏死细胞的试剂盒。单纯的PI染色能够观察DNA直方图上凋亡细胞的亚G1峰，但只能代表G0/G1期发生凋亡，无法观察S期和G2期发生的细胞凋亡，而且细胞经过固定后无法对活细胞和死细胞进行区分。Hoechst 33258可以穿透细胞膜，进入正常细胞和凋亡细胞与DNA结合，能在紫外线下显示蓝色荧光，而且染色后凋亡细胞荧光会比正常细胞明显增强。PI不能穿透细胞膜，对于具有完整细胞膜的正常细胞或凋亡细胞不能染色。而对于坏死细胞，其细胞膜的完整性丧失，PI可以穿透细胞膜使坏死细胞着色产生红色荧光。当使用这两种染料双染时，正常细胞主要被Hoechst 33258染成弱蓝色；当细胞被诱导发生凋亡时，凋亡细胞可被Hoechst 33258染成强蓝色，坏死细胞被PI染成强红色。
产品组分
H1492197 | Component | 100T | Storage | Quantity Per Test | H1492197A | Hoechst 33258 Staining Solution | 1 mL | -20℃, Store in the dark. | 10 μL per 0.5-1.0x10⁶ | H1492197B | Propidium iodide Staining Solution (PI) | 0.5 mL | -20℃, Store in the dark. | 5 μL per 0.5-1.0x10⁶ |
注：每个Test推荐染色的细胞数量为 0.5–1.0×10⁶ cells. . |
使用方法
1. 细胞准备
贴壁细胞：6孔板培养细胞至对数生长期，PBS清洗两次，加入1 mL的PBS
悬浮细胞：预冷PBS清洗细胞两次后，按照 0.5–1.0×10⁶ /mL的密度用PBS重悬。
2. 染色
贴壁细胞：在6孔板中加入10 μL Hoechst 33342和5 μL PI，4℃，避光孵育20-30分钟。
悬浮细胞： 取1 mL细胞悬液，加入10 μL Hoechst 33342和5 μL PI，4℃，避光孵育20-30分钟。
注：染色结束后尽快进行后续荧光检测，每个检测细胞数不超过 1.0×10⁶ 个。
3. 荧光显微镜检测与分析
贴壁细胞：弃染色液，PBS洗涤两次，加入适量PBS，然后在荧光显微镜下观察。
悬浮细胞：PBS清洗两次。用PBS重新悬浮细胞，置于培养板中观察红色和蓝色荧光。
注意事项
1. 染色完成后需要尽快进行检测。
2. Hoechst 33258和PI对人体有害，使用时请注意防护。
3. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
4. 本产品仅限于科研使用，不得用于临床诊断或治疗。
The Hoechst33258/PI Apoptosis Assay Kit is a detection kit that uses a dual fluorescence staining method with Hoechst 33258 and Propidium Iodide (PI) to analyze cell cycle and cell necrosis. While PI staining alone can observe the sub-G1 peak of apoptotic cells on the DNA histogram, it can only represent apoptosis in the G0/G1 phase and cannot observe apoptosis in the S and G2 phases. Moreover, after fixation, it is impossible to distinguish between live and dead cells. Hoechst 33258 can penetrate the cell membrane and bind to DNA in both normal and apoptotic cells, showing blue fluorescence under ultraviolet light. After staining, the fluorescence of apoptotic cells is significantly enhanced compared to normal cells. PI cannot penetrate the cell membrane and cannot stain normal or apoptotic cells with intact cell membranes. However, for necrotic cells, where the integrity of the cell membrane is lost, PI can penetrate the cell membrane and color the necrotic cells to produce red fluorescence. When these two dyes are used for double staining, normal cells appear weak blue, apoptotic cells appear bright blue, and necrotic cells appear bright blue and red.
Component
H1492197 | Component | 100T | Storage | Quantity Per Test | H1492197A | Hoechst 33258 Staining Solution | 1 mL | -20℃, Store in the dark. | 10 μL per 0.5-1.0x10⁶ | H1492197B | Propidium iodide Staining Solution (PI) | 0.5 mL | -20℃, Store in the dark. | 5 μL per 0.5-1.0x10⁶ |
Note: The recommended number of cells to stain per test 0.5–1.0×10⁶ cells. |
Usage method:
1. Cell Preparation
Adherent cells: Grow cells in 6-well plates to the logarithmic growth phase. Wash twice with PBS and add 1 mL of PBS.
Suspension cells: Wash cells twice with pre-cooled PBS and resuspend in PBS at a density of 0.5–1.0×10⁶ cells/mL.
2. Staining
Adherent cells: Add 10 µL of Hoechst 33342 and 5 µL of PI directly to each well of the 6-well plate. Incubate at 4 °C in the dark for 20–30 minutes.
Suspension cells: Take 1 mL of the cell suspension, add 10 µL of Hoechst 33342 and 5 µL of PI, and incubate at 4 °C in the dark for 20–30 minutes.
Note: After staining, proceed with fluorescence detection as soon as possible. The number of cells for each detection should not exceed 1×10⁶.
3. Fluorescence Microscopy Detection and Analysis
Adherent cells: Remove the staining solution, wash twice with PBS, add an appropriate amount of PBS, and observe under a fluorescence microscope.
Suspension cells: Wash twice with PBS, resuspend the cells in PBS, transfer to a culture dish, and observe for red and blue fluorescence.
Matters needing attention:
1. After the staining process is completed, the detection should be carried out as soon as possible.
Hoechst 33258 and PI are harmful to the human body. Please take protective measures when using them.
2. For your safety and health, please wear a laboratory coat and put on disposable gloves when operating.
3. This product is exclusively for scientific research purposes and must not be used for clinical diagnosis or treatment.
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