细胞周期（cell cycle）是指细胞从一次分裂完成开始到下一次分裂结束所经历的全过程，细胞周期可以分为四个时相：G1(DNA 合成前期)；S 期 （DNA 合成期）；G2 期（DNA 合成后期）和M 期（有丝分裂期）。根据染色体的倍数进行分类分为三个时相：G1 期、S期和 G2/M 期三个时相。7-AAD ( 7- Aminoactinomycin D)是一种双链 DNA 的荧光染料。7-AAD和双链DNA结合后可以产生荧光，并且荧光强度和双链DNA的含量成正比。细胞内的DNA染色后，可以用流式细胞仪对细胞进行DNA含量测定，然后根据DNA含量的分布情况，可以进行细胞周期分析。假设 G0/G1 期细胞的荧光强度为1，那么含有双份基因组DNA的G2/M 期细胞的荧光强度的理论值为2，正在进行 DNA 复制的S期细胞的荧光强度为 1-2 之间。 本试剂盒通常应用于培养的贴壁或悬浮细胞的细胞周期与细胞凋亡检测。如果用于组织的细胞周期与细胞凋亡检测，则必须把组织消化成单细胞状态，才可以进行检测。
A1456543 | 组分 | 20 T | 50 T | 100 T | Storage | Quantity Per Test |
A1456543 A | 染色缓冲液 | 10 mL | 25 mL | 50 mL | -20℃. | 500 μL per 1 × 10⁶ cells. |
A1456543 B | 7-AAD staining solution | 100 μL | 250 μL | 500 μL | -20℃.避光保存. | 5 μL per 1 × 10⁶ cells. |
A1456543 C | RNase A | 0.4 mL | 1 mL | 2 mL | -20℃. | 20 μL per 1 × 10⁶ cells. |
注: 每个Test推荐染色的细胞数量为 1 × 10⁶ cells.
使用方法
1. 收集细胞，预冷PBS清洗两次；按照0.1–1.0 × 10⁷的密度用预冷PBS重悬；
2. 在细胞悬液中，边震荡边逐滴加入无水乙醇，使乙醇最终浓度为70%；
3. 轻柔混匀细胞，4℃过夜；
4. 预冷PBS清洗细胞两次后，按照2 × 10⁶/mL的密度用染色缓冲液重悬；
5. 每500 μL 细胞悬液中加入5 μL 7-AAD 染色液及20 μL RNase A；
6. 缓慢并充分重悬细胞，室温避光孵育30min；
7.流式检测和分析。
注：需自备PBS和无水乙醇。
注意事项
1. 荧光染料均存在淬灭问题，保存和使用过程中请尽量注意避光，以减缓荧光淬灭。
2. 7-AAD 对人体有刺激性，请注意适当防护。
3. 70%乙醇固定细胞一定要充分，否则染色不均导致结果不明显或者偏差。建议 70%乙醇-4℃固定过夜。
4. 细胞固定后要保证细胞是单细胞悬液，细胞的粘连可能会影响我们的结果。
5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
6. 本产品仅限于科研，不得用于临床诊断或治疗，不得用于食品和药品，不得存放于普通 住宅内。
7. 为了您的安全和健康，请穿实验服并戴一次性手套操作
The cell cycle refers to the complete process from the completion of one cell division to the end of the next division. The cell cycle can be divided into four phases: G1 (pre-DNA synthesis phase), S phase (DNA synthesis phase), G2 phase (post-DNA synthesis phase), and M phase (mitotic phase). Based on chromosome ploidy, it is classified into three phases: G1 phase, S phase, and G2/M phase.
7-AAD (7-Aminoactinomycin D) is a fluorescent dye that binds to double-stranded DNA. When 7-AAD binds to double-stranded DNA, it produces fluorescence, and the fluorescence intensity is proportional to the amount of double-stranded DNA present. After staining intracellular DNA, the DNA content of cells can be measured using flow cytometry. Cell cycle analysis can then be performed based on the distribution of DNA content. Assuming the fluorescence intensity of G0/G1 phase cells is 1, the theoretical fluorescence intensity of G2/M phase cells, which contain twice the genomic DNA, is 2, while the fluorescence intensity of S phase cells undergoing DNA replication ranges between 1 and 2.
This kit is commonly used for cell cycle and apoptosis detection in cultured adherent or suspension cells. If it is used for cell cycle and apoptosis detection in tissues, the tissue must first be digested into a single-cell suspension before analysis can be performed.
A1456543 | Component | 20 T | 50 T | 100 T | Storage | Quantity Per Test |
A1456543A | Dyeing buffer solution | 10 mL | 25 mL | 50 mL | -20℃. | 500 μL per 1 × 10⁶ cells. |
A1456543B | 7-AAD staining solution | 100 μL | 250 μL | 500 μL | -20℃.Store in the dark. | 5 μL per 1 × 10⁶ cells. |
A1456543C | RNase A | 0.4 mL | 1 mL | 2mL | -20℃. | 20 μL per 1 × 10⁶ cells. |
Note: The recommended number of cells to stain per test is 1 × 10⁶ cells
Instructions for use
1.Collect cells and wash twice with ice-cold PBS; resuspend in ice-cold PBS at a density of 0.1–1.0 × 10⁷ cells/mL.
2.In the cell suspension, anhydrous ethanol was added dropwise while shaking until the final ethanol concentration reached 70%.
3.Mix gently and fix overnight at 4 °C.
4.Wash cells twice with ice-cold PBS, then resuspend in dyeing buffer solution of 2 × 10⁶ cells/mL.
5.Add 5 µL of 7-AAD staining solution and 20 µL RNase A to 500 µL of the cell suspension.
6.Resuspend cells gently and thoroughly, then incubate at room temperature for 30 min in the dark.
7.Analyze samples by flow cytometry.
Note: Please bring your own PBS and absolute ethanol.
Precautions
1. Fluorescent dyes all have the problem of quenching. Please try to avoid light during storage and use to slow down fluorescence quenching.
2. Iodophor is irritating to the human body. Please take appropriate protective measures.
3. When fixing cells with 70% ethanol, make sure it is thorough; otherwise, uneven staining will result in unclear or inaccurate results. It is recommended to fix the cells overnight at -4℃ with 70% ethanol.
4. After cell fixation, ensure that the cells are in a single-cell suspension; cell adhesion may affect our results.
5. For your safety and health, please wear laboratory coats and disposable gloves during operation.
6. This product is only for scientific research and cannot be used for clinical diagnosis or treatment, nor for food or medicine. It must not be stored in ordinary residences.
7. For your safety and health, please wear laboratory coats and disposable gloves during operation.
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