Invitrogen™ eBioscience™ Propidium Iodide Staining Solution

Catalog No.	Quantity
50-187-86	2 mL

Catalog No. 50-187-86 Supplier Invitrogen™ Supplier No. 00699050

Description

Ready-to-use solution for the exclusion of nonviable cells in flow cytometric analysis. Propidium Iodide binds to double-stranded DNA by intercalating between basepairs, but is excluded from cells with intact plasma membranes.

• 5μL (40 ng)/test
• Can be used in FL3 for inviability exclusion, but should be analyzed in FL2 when used as a counterstain for FITC Annexin V

Specifications

• Color: Red
• Detection Method: Fluorescence
• For Use With (Application): Viability Assay
• For Use With (Equipment): Flow Cytometer
• Product Type: Staining Solution
• Dye Type: Cell-Impermeant
• Emission: 535/617
• Product Line: eBioscience
• Quantity: 2 mL

Frequently Asked Questions (FAQs)

Is annexin V specific for any particular species?

Annexin V binding of phospholipids is not species-specific. In the presence of millimolar concentrations of Ca2+, Annexin V binds to several phospholipids, but exhibits the highest affinity for phosphatidylserine. This feature is useful for screening cells undergoing apoptosis. Phosphatidylserine is usually distributed on the inner leaflet of the lipid bilayer, but when cells start to undergo apoptosis, the phosphatidylserine moves to the outer leaflet. The translocation of phosphatidylserine to the outer leaflet makes it available for Annexin V staining. Normal, intact cells will not stain with Annexin V, while cells undergoing apoptosis will be.

Annexin V staining can be visualized by fluorescence microscopy or flow cytometry using conjugates such as Annexin V-FITC, or Annexin V-biotin in conjugation with a streptavidin-conjugated fluorophore. It is important to note that experiments with Annexin V require that the cells under study are live cells with intact membranes. It is not suitable for use with fixed cells as the membrane develops holes during the fixation process, making the phosphatidylserine on the inner face of the membrane accessible for Annexin V binding. For similar reasons, this product is also not suitable for use with tissue sections. Because necrotic cells also have numerous membrane breaches, these cells are also often stained with Annexin V. A quick way to differentiate apoptotic from necrotic cells is with a double staining regime using Annexin V-FITC (green fluorescence) and the DNA staining dye propidium iodide (red fluorescence). Propidium iodide requires membrane breaches in order to gain access to the cell nucleus.

Using the Annexin V-FITC/propidium iodide double staining regime, the following staining patterns are observed with apoptotic and necrotic cells:
1. Cells that are normal will not stain with either Annexin V-FITC or propidium iodide (indicating no phosphatidylserine on the surface and no holes in the membrane).
2. Cells that are starting to undergo apoptosis will stain with Annnexin V-FITC only (indicating phosphatidylserine on the surface but no holes in the membrane).
3. Cells that are in late apoptosis and necrotic cells will stain with both Annexin V-FITC and with propidium iodide (indicating that we cannot tell where the phosphatidylserine is located and that there are holes in the membrane).

Phosphatidylserine is an ubiquitous membrane component among mammalian species; therefore, we believe that Annexin V has utility in monitoring apoptosis with any mammalian-derived cell line. One report (O'Brien et al., 1997) has even indicated its utility in monitoring apoptosis with plant cells. Dive, C., C.D. Gregory, D.J. Phipps, D.L. Evans, A.E. Milner, and A.H. Wyllie (1992) Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochem. Biophys. Acta 1133:275-285. Koopman, G., C.P.M. Reutlingsperger, G.A.M. Kuitjen, R.M.J. Keehnen, S.T. Pals, and M.H.J. van Oers (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415-1420. O'Brien, I.E., C.P. Reutelingsperger, and K.M. Holdaway (1997) Annexin-V and TUNEL use in monitoring the progression of apoptosis in plants. Cytometry 29(1):28-33.

How do I discriminate between live cells and dead cells when doing a flow cytometry analysis?

Discrimination between viable and non-viable cells can be carried out with the use of the 7-AAD Viability Dye (Cat. No. 00-6993-50) or Propidium Iodide (PI) Staining Solution (Cat. No. 00-6990-50). The 7-AAD or PI will mark the non-viable cells by binding to the nuclei of those cells. The nucleic acid of viable cells will not be accessible to the dye and will not be stained. When analyzing the data collected, gate out all cells stained with the viability dye. When staining for intracellular proteins, use the Fixable Viability Dye eFluor 455UV, 450, 520, 660, or 780.

For Research Use Only. Not for use in diagnostic procedures.