What is in annexin V binding buffer?

The Annexin V Binding Buffer is a 10X concentrate composed of a 0.2 µm sterile filtered 0.1M Hepes (pH 7.4), 1.4M NaCl, and 25 mM CaCl2 solution. Prior to staining cells, an appropriate quantity of a 1X working solution should be made by diluting the 10X concentrate 1:10 with distilled water.

How does annexin V work?

Annexin V, a 36-kDa calcium-binding protein, binds to PS; therefore, fluorescently labeled Annexin V can be used to detect PS that is exposed on the outside of apoptotic cells. Annexin V can also stain necrotic cells because these cells have ruptured membranes that permit Annexin V to access the entire plasma membrane.

How do you titrate annexin V?

Using flow cytometry, titrate annexin V against the usual number of cells you’ll be using in your experiments, at the appropriate volume. Typical values are 0.2-0.3 × 106 cells in 200 μL of BB. Use 2.5 mM calcium, and determine the best concentration of annexin V per 100 μL.

What is in binding buffer?

The binding buffer is an isotonic buffer containing calcium, which is essential for the binding of Annexin V to phosphatidylserine.

How do you make a binding buffer?

2 L + 80 ml Binding buffer BB

  1. Weigh out 955.2 g GuHCl and add to a 2 L beaker.
  2. Add 500 mL freshly drawn milliQ . Stir and heat to 60 °C to dissolve.
  3. Once dissolved, make up to 2 L with Isopropanol (approx. 800 mL ) using a 1 L graduated cylinder.
  4. Add 80 mL 3M sodium acetate , pH 5.2 .
  5. Add 1 mL Tween-20 .

What is annexin V assay?

The annexin V binding assay is a powerful tool for the quantification of apoptosis and for distinguishing between apoptotic and necrotic cells and is used by many laboratories.

What is Pi in flow cytometry?

Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs. PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm.

What is the function of binding buffer in DNA extraction?

The chaotropic salt binding buffer allows the highest DNA binding of any column method. Powerful wash buffers remove all traces of protein and salt. DNA is eluted in a low-salt buffer to allow for pH stabilization of the DNA in storage.

What is binding buffer used for?

DNA Binding Buffer is designed for use in the DNA Clean & Concentrator family of products. This buffer facilities efficient binding and recovery of DNA from enzymatic reactions and impure DNA samples.