吖啶橙(Acridine Orange, AO)是一种异染性荧光染料，其荧光颜色因结合物不同而变化：与双链DNA结合：嵌入碱基对间，激发后发射绿色荧光（Ex 488nm, Em 530nm）。与单链RNA或溶酶体结合：静电吸附，发射橙红色荧光（Em＞640nm）。在荧光显微镜下观察，吖啶橙可透过正常细胞膜，使细胞核呈绿色或黄绿色均匀荧光；而在凋亡细胞中，因染色质固缩或断裂为大小不等的片断，形成凋亡小体。吖啶橙使其染上致密浓染的黄绿色荧光，或黄绿色碎片颗粒；而坏死细胞黄绿荧光减弱甚至消失。吖啶橙(AO)常与碘化丙啶(PI)合用双染，可区分出正常细胞、凋亡细胞及坏死细胞。
产品组份
A1456513 | Component | 100 Test | 500 Test | Storage Condition | Quantity Per Test | A1456513A | AO Dilution Buffer | 10 mL | 50 mL | 2-8℃ | 0.1 mL per 0.5-1.0 × 10⁶ cells | A1456513B | AO Staining Solution | 100 μL | 500 μL | 2-8℃, Protect from light. Do not freeze | 1 μL per 0.5-1.0 × 10⁶ cells |
注: 每个Test推荐染色的细胞数量为 0.5-1.0 × 10⁶ cells. |
1. 吖啶橙染色液的配制
a. 使用前用双蒸水将AO 稀释缓冲液稀释10倍；
b. 将AO染色液与AO 稀释缓冲液 1:1000的比例混合，配制成吖啶橙染色液。例如取10 μL的AO染色液加入10mL的AO 稀释缓冲液中，即得10mL的吖啶橙染色液。
2. 吖啶橙染色
a. 对于贴壁细胞：
(a) 轻轻吸除孔板中的培养液，PBS洗涤约10秒，吸除PBS。
(b) 加入吖啶橙染色液，室温染色5分钟，然后吸除染色液，PBS洗涤约10秒，重复一次。
注：对于在六孔板中培养、汇合度超过80%的贴壁细胞，建议按1 mL/well 加入染色工作液，也可根据具体的实验体系进行优化。
(c) 加入适量细胞培养液染色缓冲液或其它适当溶液覆盖孔板底部，置于显微镜下观察。可根据检测需求，在 Ex/Em = 488/530 nm 下观察绿色荧光，在 Ex/Em = 540/640 nm 下观察红色荧光，也可以使用荧光酶标仪采用其底读功能进行荧光强度检测。
b. 对于悬浮细胞：
(a) 取1mL细胞悬液，500×g室温离心5分钟，轻轻吸除培养基后加适量PBS重悬，500×g室温离心5分钟，去除PBS。
(b) 加适量吖啶橙染色液，使细胞密度约为 10⁶ 个/mL。
(c) 室温染色5分钟。
(d) 直接滴加于载玻片上，加盖盖玻片，置于显微镜下观察。可根据检测需求，在 Ex/Em = 488/530 nm 下观察绿色荧光，在 Ex/Em = 540/640 nm 下观察红色荧光，也可以在染色结束后直接进行流式细胞仪分析检测或使用荧光酶标仪检测细胞的荧光。
注：离心去除染色液能适当降低荧光背景。悬浮细胞或悬浮状态的贴壁细胞染色时所需AO染色液浓度可以考虑适当降低2-5倍，染色时间可以考虑缩短至2分钟。
注意事项
1. AO 染色液有一定毒性，请小心操作。
2. 为了您的安全和健康，请穿实验服并戴一次性手套。
3. 荧光染料存在淬灭的问题，建议染色后尽量当天完成检测。
Acridine Orange (AO) is a metachromatic fluorescent dye whose emission color varies depending on the target it binds to:
l When binding to double-stranded DNA: It intercalates between base pairs and emits green fluorescence upon excitation (Ex 488 nm, Em 530 nm).
l When binding to single-stranded RNA or lysosomes: It attaches via electrostatic interactions and emits orange-red fluorescence (Em＞640 nm).
Under a fluorescence microscope, Acridine Orange permeates the membranes of normal cells, staining the nucleus with uniform green or yellow-green fluorescence. In apoptotic cells, due to chromatin condensation and fragmentation into apoptotic bodies, AO stains them with intense, condensed yellow-green fluorescence or fragmented yellow-green particles. In necrotic cells, the yellow-green fluorescence is reduced or absent.
Acridine Orange is often used in combination with Propidium Iodide (PI) for dual staining. Since PI stains only dead cells, producing orange-red fluorescence, this method allows differentiation among normal, apoptotic, and necrotic cells.
Components
A1456513 | Component | 100 Test | 500 Test | Storage Condition | Quantity Per Test | A1456513A | AO Dilution Buffer | 10 mL | 50 mL | 2-8℃ | 0.1 mL per 0.5-1.0 × 10⁶ cells | A1456513B | AO Staining Solution | 100 μL | 500 μL | 2-8℃, Protect from light. Do not freeze | 1 μL per 0.5-1.0 × 10⁶ cells |
Note: The recommended number of cells to stain per test is 0.5-1.0 × 10⁶ cells. |
Procedure
1. Preparation of Acridine Orange Staining Solution
Mix the AO Staining Solution with the Dilution Buffer at a ratio of 1:1000 to prepare the working solution. For example, add 10 μL of AO Staining Solution to 10 mL of Dilution Buffer to obtain 10 mL of Acridine Orange staining solution.
2. Staining with Acridine Orange
a. For adherent cells:
(a) Gently aspirate the culture medium from the plate. Rinse with PBS for about 10 seconds, then remove PBS.
(b) Add Acridine Orange staining solution and incubate at room temperature for 5 minutes. Remove the staining solution and rinse with PBS for about 10 seconds. Repeat the rinse once.
Note: For adherent cells cultured in a 6-well plate with a confluence exceeding 80%, it is recommended to add the staining working solution at a volume of 1 mL per well. This volume can be optimized based on the specific experimental system.
(c) Add an appropriate amount of cell culture medium, staining buffer, or other suitable solution to cover the well bottom. Observe under a microscope. Depending on the detection requirements, green fluorescence can be observed at Ex/Em = 488/530 nm, and red fluorescence can be observed at Ex/Em = 540/640 nm. Alternatively, measure fluorescence intensity using a fluorescence microplate reader with bottom-reading capability.
b. For suspension cells:
(a) Take 1 mL of cell suspension. Centrifuge at 500×g for 5 minutes at room temperature. Gently aspirate the medium, resuspend in PBS, and centrifuge again at 500×g for 5 minutes. Remove PBS.
(b) Add an appropriate amount of Acridine Orange staining solution to achieve a cell density of approximately 10⁶ cells/mL .
(c) Incubate at room temperature for 5 minutes.
(d) A drop of the sample was directly applied onto a glass slide, covered with a coverslip, and examined under a microscope. Depending on the detection requirements, green fluorescence can be observed at Ex/Em = 488/530 nm, and red fluorescence can be observed at Ex/Em = 540/640 nm. Alternatively, after staining, analyze directly by flow cytometry or measure fluorescence with a microplate reader.
Note: Centrifugation to remove staining solution can reduce background fluorescence. For suspension cells or adherent cells in suspension, consider reducing the AO staining solution concentration by 2–5 times and shortening the staining time to 2 minutes.
Precautions
1. AO Staining Solution is toxic. Handle with care.
2. For your safety and health, wear a lab coat and disposable gloves.
3. Fluorescent dyes are susceptible to quenching. It is recommended to complete detection on the same day after staining.
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