AO/PI Double Staining Kit - Suitable for Immunofluorescence(IF),BioReagent,Biological Stain,for microscopy,for fluorescence analysis,for cell culture, high purity

Cat. No.: A1492198

GRADE & PURITY BioReagent, Biological Stain, for Fluorescence analysis, for Microscopy, for Cell culture, Suitable for Immunofluorescence(IF)

Overview

The AO/PI Double Staining Kit is a detection system based on fluorescent nucleic acid dyes, designed to quickly and reliably distinguish live, apoptotic, and necrotic cells in biological samples. Its core principle utilizes the differences in cell membrane permeability and the specific fluorescent reactions upon binding to nucleic acids of the two dyes, Acridine Orange (AO) and Propidium Iodide (PI).

AO can penetrate the cell membranes of all cell types (live, apoptotic, and necrotic). After entering the cell, AO binds to nucleic acids, but its fluorescence color depends on the target: when it binds to double-stranded DNA (dsDNA) in the nucleus, it emits green fluorescence (Ex 488 nm, Em 530 nm); whereas when it binds to single-stranded DNA (ssDNA) or RNA, it emits red fluorescence (Em >640 nm). In contrast, PI cannot penetrate cells with intact cell membranes. It only enters late apoptotic and necrotic cells, whose cell membranes have been compromised, and binds to DNA to produce red fluorescence (Ex 535 nm, Em 617 nm).

Therefore, after AO/PI double staining, observation under a fluorescence microscope allows clear differentiation of cells in different states: live cells display normal green fluorescence; early apoptotic cells show bright green speckled nuclear morphology (only AO enters); late apoptotic and necrotic cells appear orange-red or strong red fluorescence due to the combined effect of AO and PI.

Procedure

1. Preparation of Working Solution

(1) Take out the reagents and allow them to reach room temperature. Dilute the AO/PI Dilution Buffer 10-fold with double-distilled water before use.

(2) Prepare the AO/PI Staining Working Solution: The concentration of AO stock solution in this product is 10 mg/mL, and PI stock solution is 1 mg/mL . Add 50 μL of PI stock solution and 10 μL of AO stock solution to 10 mL of diluted AO/PI Dilution Buffer. Vortex to mix well, resulting in 10 mL of AO/PI Staining Working Solution.

Note: Since optimal staining conditions may vary with cell type, it is recommended that users determine the appropriate dye concentration individually based on practical conditions.

2. Staining

a. For Adherent Cells:

(a) Seed cells in culture dishes, multi-well plates, or on coverslips, and treat the cells as required by the experimental design. Gently aspirate the medium from the wells and wash with PBS for about 10 seconds, then remove the PBS.

(b) Add an appropriate volume of AO/PI Staining Working Solution, and gently swirl to ensure the dye evenly covers all cells.

Note: For adherent cells cultured in 6-well plates with a confluency over 80%, it is recommended to add 1 mL of staining working solution per well. This can be optimized according to the specific experimental setup.

(c) Incubate the cells at room temperature, protected from light, for 1-10 minutes.

(d) Aspirate the staining solution, wash 2-3 times with PBS, then add serum-free culture medium. Observe immediately under a fluorescence microscope: observe AO-DNA green fluorescence at Ex/Em = 488/530 nm, and PI-DNA red fluorescence at Ex/Em = 535/617 nm. Alternatively, analysis by flow cytometry or detection with a fluorescence microplate reader can be performed after staining.

b. For Suspension Cells:

(a) After treating the cells as required by the experimental design, count the cells. Take an appropriate number of cells, centrifuge at 500 × g for 5 minutes at room temperature, gently aspirate the medium, resuspend in an appropriate amount of PBS, and centrifuge again at 500 × g for 5 minutes at room temperature to remove the PBS.

(b) Add an appropriate amount of AO/PI Staining Working Solution to achieve a cell density of approximately 10⁶ cells/mL.

(c) Incubate the cells at room temperature, protected from light, for 1-10 minutes.

(d) Place a drop directly on a glass slide, cover with a coverslip, and observe under a microscope: observe AO-DNA green fluorescence at Ex/Em = 488/530 nm, and PI-DNA red fluorescence at Ex/Em = 535/617 nm. Alternatively, analysis by flow cytometry or detection with a fluorescence microplate reader can be performed after staining.

Note: If background interference is significant, centrifuge to remove the staining solution, wash 1-2 times with PBS, and then observe under the microscope.

Precautions

1.    AO/PI staining solutions are somewhat toxic; handle with care .

2.    For your safety and health, please wear a lab coat and disposable gloves.

3.    Fluorescent dyes are subject to quenching; it is recommended to complete detection on the same day after staining whenever possible.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

Contents & Storage

A1492198	Components	100T	500T	Storage	Quantity Per Test
A1492198A	AO Staining Solution	100 μL	500 μL	2-8℃, Store in the dark.	1 μL per 0.5-1.0 × 10⁶ cells
A1492198B	PI Staining Solution	500 μL	2500 μL	-20℃. Store in the dark.	5 μL per 0.5-1.0 × 10⁶ cells
A1492198C	AO/PI Dilution Buffer	10 mL	50 mL	2-8℃	0.1 mL per 0.5-1.0 × 10⁶ cells

Note: The recommended number of cells to stain per test is 0.5-1.0 × 10⁶ cells.

Images

AO/PI Double Staining Kit (A1492198)

AO/PI Double Staining Kit (A1492198)

HeLa cells were treated with camptothecin (C111281) and subsequently stained using an AO/ PI double-staining kit (A1492198). The staining was performed by incubating the cells with AO (1:1000 dilution) and PI (1:200 dilution) for 10 minutes at room temperature. Cell imaging was conducted using a confocal microscope with the following settings: AO was excited at 488 nm and its emission was collected at 530 nm, while PI was excited at 535 nm with an emission at 617 nm. Analysis of the merged fluorescence images identified three distinct cell populations based on their differential staining:Viable Cells (Green): These cells exhibit green fluorescence, resulting from AO uptake and binding to DNA. Their intact plasma membrane excludes PI.Dead Cells (Red): These cells display bright red fluorescence because their compromised membranes allow PI to enter, bind to DNA, and dominate the fluorescence signal.Late Apoptotic Cells (Orange/Yellow): This population is characterized by a yellow-orange signal. This intermediate phenotype arises from the co-localization of AO (green) and PI (red) fluorescence, indicating increased membrane permeability that permits PI influx while some AO signal remains.(Optional addition for completeness): It is noteworthy that early apoptotic cells, which are not explicitly mentioned here, would typically exhibit bright green or condensed green nuclei due to chromatin condensation and still maintain membrane integrity enough to exclude PI.