Biolegend, 422201, Annexin V Binding Buffer, 100ml

Annexin V Binding Buffer has been formulated for flow cytometric labeling of apoptotic cells with Annexin V reagents.  The buffer is provided as a ready-to-use solution.
100ml
Storage & Handling: Store between 2°C and 8°C.
Application: FC - Quality tested
Recommended Usage: Annexin V Binding Buffer is recommended for use with Annexin V reagents. Cells should be resuspended in Annexin V Binding Buffer at a concentration 1x106/ml and then combined with Annexin V reagents at the recommended volumes.
Application Notes: Annexin V Staining Wash cells twice with cold BioLegend Cell Staining Buffer (Cat. No. 420201) and then resuspend cells in Annexin V Binding Buffer (Cat. No. 422201) at a concentration of 1x106 cells/mL. Transfer 100 µL of cell suspension in 5 mL test tube. Add 5 µL of fluorochrome conjugated Annexin V. Stain with a viability dye, such as PI (Cat. No. 421301), 7-AAD (Cat. Nos. 420403 & 420404), or Helix NP dyes (Cat. Nos. 425301, 425303, & 425305), if desired. Gently vortex the cells and incubate for 15 min at RT (25°C) in the dark. Add 400* µL of Annexin V Binding Buffer (Cat. No. 422201) to each tube. *For more concentrated samples, add a minimum of 200 µl of Annexin V Binding Buffer in this step. Analyze by flow cytometry. For a better experience detecting apoptosis, we now recommend Apotracker™. Cell staining with Apotracker™ is Calcium independent. Thus, no special buffers are required, and the protocol can be shortened for single-step co-staining with other reagents.
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Gene ID: NA
Regulatory Status: RUO
Other Names: Annexin-V-Binding Buffer
Q: How is your Annexin made and what sequence does it cover?
A: It is made in E. coli, covering human aa Met1-Asp320.
Q: How does pH and staining temperature affect Annexin V-Phosphatidylserine binding?
A: Annexin-Phosphatidylserine binding is lost below pH 5.2 and with prolonged incubation over a temperature of 42°C.
Q: Why do I need to use Annexin V Binding Buffer?
A: Annexin V binding requires the presence of calcium in the solution.  So, we provide Annexin V Binding Buffer (cat # 422201), which is optimized for the best performance of Annexin V staining.
Q: Can I use RPMI during Annexin V staining?
A: It is best to follow protocol as described on the product data sheet. Moreover, RPMI 1640 has a relatively high concentration of phosphate and low calcium ion concentration, which negatively impacts Annexin binding to its target phosphatidylserine (PS). Measurement of cell death by using Annexin V may also be significantly affected by time of incubation on ice, calcium concentration, and type of medium.
Q: Can I freeze Annexin V conjugates?
A: It should not be frozen as it will lead to loss of biological activity due to dimerization.
Q: Is Annexin V suitable for conjugation with the Maxpar® kit for CyTOF®?
A: Maxpar® Labeling kits require the protein to be partially reduced, so the metal chelate can be introduced through an SH group in the hinge region of the reduced antibody. Human Annexin V contains only one Cysteine which was reported to be chemically inactive. Thus, the Maxpar® labeling protocol would not work with Annexin V, unless a free –SH group can be introduced to Annexin V.  For more information regarding SH-mediated conjugation of Annexin V please consult published papers such as this one.