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
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Cat.
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Products
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50 Tests
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100 Tests
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200 Tests
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500 Tests
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Storage
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E-CK-A163
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DAPI Reagent (25μg/mL)
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250 μL
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500 μL
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1 mL
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1.25 mLx2
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2~8°C
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Manual
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One Copy
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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.
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