Annexin V-FITC Apoptosis Detection Kit Description 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 Components Contents Cat#:B32115 Cat#:B32117 Annexin V-FITC 250 µl 250 µl × 2 PI Staining Solution 250 µl 250 µl × 2 1 × Binding Buffer 25 ml 25 ml × 2 Storage Store in refrigerator (2–8° C) and protect from light. Notice 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. Protocol 1. Induce apoptosis in cells using the desired method. Prepare a negative control by incubating cells in the absence of inducing agent. 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. Order & Inquiry Order & Inquiry Tel: (713)732-2181 Fax: +1-866-747-4781 E-mail: [email protected] Tel: +49-89-46148500 Fax: +49-89-461485022 E-mail: [email protected]