Annexin V-FITC Apoptosis Detection Kit letter

Annexin V-FITC Apoptosis Detection Kit
Annexin V-FITC kit provides a rapid and convenient assay for
apoptosis. Annexin V-FITC kit detects the externalization of
phosphatidylserine in apoptotic cells using recombinant annexin V
conjugated to green-fluorescent fluorescein isothiocyante (FITC) dye
and necrotic cells using propidium iodide (PI) by flow cytometry and
fluorescence microscope. The AnnexinV-FITC kit uses annexin V
conjugated with FITC to label phosphatidylserine sites on the
membrane surface. The kit also includes propidium iodide (PI) stains
necrotic cells with red fluorescence. After treatment with both probes,
apoptotic cells show green fluorescence, dead cells show red and
green fluorescence, and live cells show little or no fluorescence.
7.As soon as possible, analyze the stained cells by flow cytometry,
measuring the fluorescence emission at 530 nm (e.g., FL1) and
>575 nm (e.g., FL3). The population should separate into three
groups: live cells will show only a low level of fluorescence,
apoptotic cells show green fluorescence and dead cells show both
red and green fluorescence.Confirm the flow cytometry results by
viewing the cells under a fluorescence microscope, using filters
appropriate for fluorescein (FITC) and rhodamine (TRITC) or Texas
RedR dye.
Expected results
Annexin V-FITC
250 µl
250 µl × 2
PI Staining Solution
250 µl
250 µl × 2
1 × Binding Buffer
25 ml
25 ml × 2
Store in refrigerator (2–8° C) and protect from light.
Please centrifuge before use !
Figure 1.Jurkat cells (T-cell leukemia, human) treated with 0, 4, 8, 16 ng/ml TNF
for 24 hours. Cells were then treated with the reagents in the kit, followed by flow
cytometric analysis. Note that the TNF-treated cells have a higher percentage of
apoptotic cells than the basal level of apoptosis seen in the control cells.
1. Induce apoptosis in cells using the desired method. Prepare a
negative control by incubating cells in the absence of inducing
2. Harvest the cells after the incubation period and wash in cold
phosphate-buffered saline(PBS).
3. Re-centrifuge the washed cells, discard the supernatant, and
resuspend the cells in 100 µl 1 × Binding Buffer.
4. Add 5 µL Annexin V -FITC and 5 µL PI Staining Solution to each
100 µL of cell suspension.
5. Incubate the cells at room temperature for 15 minutes.
6. After the incubation period, add 400 µL of 1 × Binding Buffer, mix
gently and keep the samples on ice.
Figure 2. Apoptosis of 3T3 cells induced by high dose of H2O2.
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