Wednesday, March 4, 2015

Product Focus: Annexin V-FITC Kit

Benefits

  • Detects apoptosis earlier in the process than DNA-based assays (TUNEL).
  • Rapid labeling of cells. Cell staining takes only 10 minutes.
  • No cell fixation or processing required, reducing the detection time and allowing the cells to be used for further study.
  • PI is included with the kit to differentiate apoptotic cells from viable and necrotic cells.


Introduction

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.

Catalogue B32115 (50 rxns) B32117 (100 rxns)
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.


Experimental 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.
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.

Biotool™ Annexin V-FITC Apoptosis Detection Kit Protocol



Where can I find more information about Biotool?

Visit the manufacturer page at www.stratech.co.uk/biotool, email info@stratech.co.uk or call +44 (0) 1638 782600.

Stratech Scientific is a distributor of high quality, competitively priced, reliable products for research laboratories throughout the UK and Europe. Please contact us to find out which ranges we can supply in your country.


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