Introduction
Elabscience® 7-AAD Reagent (100μg/mL) is developed to identify apoptotic and necrotic
cells.
7-AAD (7-amino-actinomycin
D) 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, 7-AAD can enter late apoptotic or
necrotic cells to stain DNA. Cells at different apoptotic stages can be
distinguished by using 7-AAD and Annexin V.
Components
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产品编号
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产品名称
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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-A162
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7-AAD Reagent (100μg/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 mL × 2
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2~8°C, shading light
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Jurkat cells were treated with 5 μM
Camptothecin and detected by this reagent and Annexin V-APC.
Jurkat cells were treated with 5 μM Camptothecin (Right) or not (Left) for 4 h. Annexin V-APC single-positive cells were early apoptotic
cells, Annexin V-APC and 7-AAD double-positive cells were necrotic or late
apoptotic cells, and PI single-positive cells were nude nuclear cells.
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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.
Note: 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 7-AAD Reagent (100μg/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.
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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