Introduction

Elabscience^®  DAPI Reagent (25μg/mL) is developed to identify apoptotic and necrotic cells.

DAPI Reagent has a high DNA binding constant and is efficiently excluded by intact cells. It is useful for DNA analysis and dead cell discrimination during flow cytometric analysis. Due to the loss of integrity of membrane, DAPI can enter late apoptotic or necrotic cells to stain DNA. Cells at different apoptotic stages can be distinguished by using DAPI and Annexin V.

Components

Jurkat cells were treated with 5 μM Camptothecin and detected with this reagent and Annexin V-APC.

Jurkat cells were cultured with (Right) or without (Left)  5 μM Camptothecin for 4 h. Annexin V-APC single-positive cells were early apoptotic cells, Annexin V-APC and DAPI double-positive cells were necrotic or late apoptotic cells, and DAPI single-positive cells were naked nuclei.

Staining Procedure

1. Induce apoptosis of suspension cells with reagents of interest. Collect cell cultures, centrifuge at 300 g for 5 min and discard the supernatant. Add PBS to wash the cells and resuspend the cells gently followed by the cell counting.

Tip: This product is only validated in suspension cells. Good cell viability is the key to the experiment. When the adherent cells are used for apoptotic detection, treatments like digestion may increase the ratio of necrotic or apoptotic cells and cause uncontrollable effects on the experimental results. Please be aware！

2. Split the cell suspension into tubes, 1~5 × 10⁵ cells for each, centrifuge at 300 g for 5 min and discard the supernatant. Add PBS to wash the cells and discard the supernatant. Add 500 μL of 1 × Annexin V Binding Buffer[E-CK-A151] to resuspend the cells.

3. Add 5 µl of Annexin V-APC[E-CK-A117] and 5 µl of  DAPI Reagent (25μg/mL) to each tube.

4. Gently vortex the cells and incubate at room temperature for 15~20 min in the dark.

5. Analyze the cells immediately with proper machine settings. Otherwise, place the cells on ice in the dark and analyze within 1 h.

Storage

Store at 2~8°C for one year in dark.  DAPI Reagent (25μg/mL) should be spilt into small tubes.

Cautions

1. For maximal assay performance, this reagent should be used within 12 months. Avoid freeze / thaw cycles.

2. Detect apoptosis as soon as possible after staining to avoid the increase number of apoptosis or necrosis.

3. Avoid extended exposure of the samples to direct light to protect the fluorophores from quenching.

4. For your safety and health, please wear the lab coat and disposable gloves before the experiments.

Citations