Annexin V:

In normal viable cells, phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. However, in the intermediate stages of apoptosis, PS is translocated from the inner to the outer leaflet of the membrane, exposing PS to the external cellular environment where it can be detected. Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine. In flow cytometry, annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine, a marker of apoptosis when it is on the outer leaflet of the plasma membrane.

 

Propidium Iodine:

Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability or DNA content in cell cycle analysis. Propidium Iodide cannot cross the membrane of live cells, making it useful to differentiate necrotic, apoptotic and healthy cells. PI also binds to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA staining.Propidium iodide (PI) is a popular red-fluorescent nuclear and chromosome counterstain. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population.

REAGENT PREPARATION

 

•Dilute the 10X concentrated Binding Buffer 10 fold with distilled water and place the diluted buffer on ice. Prepare a quantity sufficient for the expected number of assays.

•Dissolve the 250 μg PI in 1 mL of 1X Binding Buffer and place the PI solution on ice.

•After use, the solutions should be stored at 2 – 8°C

PROCEDURE FOR STAINING

  1. Induce apoptosis by desired method.

  2. Wash cell samples with ice-cold PBS and centrifuge for 5 minutes at 500 x g at 4°C. Discard supernatant, and resuspend the cell pellets in ice-cold 1X Binding Buffer to 5 x 105~ 5 x 106 cells / mL. Keep tubes on ice.

  3. Add 1 μL of annexin V-FITC solution and 5 μL of dissolved PI to 100 μL of the cell suspensions prepared as given in step 1. Mix gently.

  4. Keep tubes on ice and incubate for 15 minutes in the dark.

  5. Add 400 μL of ice-cold 1X binding buffer and mix gently.

  6. Analyze cell preparations within 30 minutes by flow cytometry or fluorescence microscopy

Below there are some case which shows apoptosis and necrosis in different conditions:

CASE 1 shows three picture 1 is unstain sample, 2 is fresh stain sample with annexin V and PI while the 3 is is the sample in which apoptosis induced artificially. % of apoptosis and necrosis is more in 3 sample.

 

 

 

 

 

 

CASE 2 shows three picture 1 is unstain sample, 2 is fresh stain sample with annexin V and PI while the 3 is is the sample in which apoptosis induced artificially. % of apoptosis and necrosis is more in 3 sample.

 

 

 

 

 

 

 

 

 

 

CASE 3 shows three picture 1 is unstain sample, 2 is fresh stain sample with annexin V and PI while the 3 is is the sample in which apoptosis induced artificially. % of apoptosis and necrosis is more in 3 sample.

 

 

 

 

 

 

 

 

 

 

REFERENCE:

Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH.Blood. 1994 Sep 1;84(5):1415-20.

Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells. Schutte B, Nuydens R, Geerts H, Ramaekers F.J Neurosci Methods. 1998 Dec 31;86(1):63-9.

Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (Jul 1995). "A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V". Journal of Immunological Methods. 184 (1): 39–51.

Annexin-FP488 fluorescent staining protocol at Interchim

Wen Y, Edelman JL, Kang T, Sachs G (May 1999). "Lipocortin V may function as a signaling protein for vascular endothelial growth factor receptor-2/Flk-1". Biochemical and Biophysical Research Communications. 258 (3): 713–21. doi:10.1006/bbrc.1999.0678. PMID 10329451.

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