What is 7-AAD?
7-AAD (7-Aminoactinomycin D) is a fluorescent dye used in flow cytometry to evaluate cell viability or apoptosis. It can intercalate into double-stranded DNA and fluoresces red when bound to DNA, which allows it to be used to identify dead or damaged cells. It is excluded from live cells and cells in early apoptosis, but it can penetrate cell membranes of dead or late apoptotic cells.
What is the Mechanism of 7-AAD?
7-AAD is a fluorescent dye used in flow cytometry to evaluate cell viability or cell death. The mechanism of 7-AAD involves the intercalation of this dye into DNA.
7-AAD cannot pass through the intact membranes of live cells. Therefore, only the cells with compromised membranes (dead or dying cells) can be stained with 7-AAD. Once the cell membrane integrity is lost, 7-AAD enters the cell and binds to the G-C rich regions of DNA.
Upon binding to DNA, 7-AAD experiences a spectral shift and emits highly fluorescent red light, which can be detected in flow cytometry. The intensity of the fluorescence is proportional to the amount of DNA in the cell indicating the level of cell death. Thus, 7-AAD is a useful tool for distinguishing between live and dead cells in a sample. It is also helpful in apoptosis studies and cytotoxicity assays.
7-AAD Cell Viability Flow Cytometry Protocol
7-AAD is a marker used for identifying dead cells in a population. It binds selectively and irreversibly to the GC regions of dsDNA, and it's used in flow cytometry to evaluate cell viability or cell death. Here is a simple 7-AAD staining protocol for flow cytometry:
Materials Needed:
- PBS (Phosphate Buffered Saline)
- FC (Flow cytometry) Buffer
- 7-AAD viability staining solution
- Cell samples
- BD FACS (or any brand) tubes
Step 1: Sample Preparation
1. Use healthy cells that were recently harvested and counted. Take a volume of 1-2 million cells per assay as a baseline.
Step 2: Cell Staining
1. Centrifuge the cells at 1,200 rpm (roughly 300 g) for 5 minutes.
2. Resuspend the cells in PBS or cell culture medium.
3. Add 5 µL of 7-AAD staining solution per 1 million cells.
4. Incubate the cells at room temperature for 15-20 minutes, protected from light.
Step 3: Wash and Resuspension in FC Buffer
1. Carefully add 2 mL of cold FC buffer to each tube and gently vortex.
2. Centrifuge at 1,200 rpm for 5 minutes.
3. After the spin, discard the supernatant and re-suspend the cell pellet in FC buffer at a concentration of up to 107 cells/mL.
Step 4: Flow Cytometry Analysis
1. Analyze the cells by flow cytometry within 1 hour after staining.
2. Analyze both the sample and control on the flow cytometer setting the forward, side scatter and 7-AAD channels to logarithmic.
3. Pre-set the flow cytometer detector and compensation settings using a single stained control.
4. Collect at least 10,000 gated events.
Remember: Dead cell staining using 7-AAD is irreversible, so do not use the stained samples for further experiments. Take care to avoid light exposure to samples and to the 7-AAD dye itself for best results. Also, ensure to follow manufacturer's instructions.
Note: The protocol may vary including the volume of reagents based on the type of cells used, type of experiment, levels of cell death, and the specific flow cytometer model.
