No problem! Key content: Essential steps for apoptosis operation

The detection of apoptosis can be conducted from six aspects: morphology, cell membrane, DNA, mitochondria, Caspase cascade, and protein expression. The commonly used methods are flow cytometry and TUNEL staining.

Flow cytometry (Annexin V-FITC/PI double staining)

Procedure:

Collect and process the cells (including suspended cells), and wash them twice with PBS.

Resuspend the cells in 1× Binding Buffer and adjust the density to 1×10⁶ cells/mL.

Add 5 μL Annexin V-FITC and 5–10 μL PI, and incubate at room temperature in the dark for 15 minutes.

Perform flow cytometry detection within 1 hour.

Result interpretation:

Lower right quadrant (Annexin V⁺/PI⁻): Early apoptosis

Upper right quadrant (Annexin V⁺/PI⁺): Late apoptosis/necrosis

The total apoptosis rate in the model group is usually 2–3 times higher than that in the control group (e.g., from ~10% to ~30–50%).

Figure 1.png  Figure 2.png

Tissue digestive enzymes (Qida Biotechnology, product number: SD0013)  

Cell digestion solution (Qida Biotechnology, product number: SD0003)

2. TUNEL staining

Procedure:

Place the cells on a 24-well plate with cell slides.

Fix with 4% paraformaldehyde at room temperature for 30 minutes.

Dip with TUNEL staining solution and incubate at room temperature in the dark for 1 hour.

封片 with DAPI， observe and take pictures with a laser confocal microscope.

Judgment： The model group shows obvious green fluorescence (TUNEL positive)， while the control group has almost no signal.