Sunday, May 13, 2012

Fluorescent Liposomes: ANTS/DPX Fusion Assay

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  1. In order to perform fusion assay three batches of liposomes are needed: a) ANTS encapsulated liposomes b) DPX encapsulated liposomes c)  ANTS/DPX co-encapsulated liposomes
  2. On fluorometer set the excitation monochromator to 360 nm and emission monochromator to 530 nm.
  3. For a fusion assay that utilizes 50 µM total lipid, take the appropriate amount of ANTS encapsulated liposomes and add it to the buffer inside the fluorometer cuvette to make a 25 µM of ANTS-liposomes and also  take the appropriate amount of DPX encapsulated liposomes and add it to the buffer inside the fluorometer cuvette to make a 25 µM of DPX-liposomes. The total lipid concentration will be 50 µM. Please keep in mind that we are talking about lipid concentration and not the concentration of encapsulated molecules.
  4. Measure the fluorescence of the solution and adjust it to an arbitrary unit of 100%.
  5. Take the appropriate amount of ANTS/DPX co-encapsulated liposomes and add it to the buffer inside the fluorometer cuvette to make a liposome solution with total lipid concentration of 50 µM (twice the lipid concentration of ANTS encapsulated liposomes).
  6. Measure the fluorescence of the solution and adjust it to an arbitrary unit of 0%.  This represents the theoretical fusion product (fully quenched) of all ANTS encapsulated liposomes and DPX encapsulated liposomes.
  7. Add the proper fusogen, such as protons, fusogenic peptides or divalent cations and measure the fluorescence. Make sure that the solution inside the cuvette is under constant stirring.
  8. Liposome fusion results in the decrease of fluorescence due to quenching of ANTS by DPX. DPX is not a fluorescent molecule and it is the collisional quencher of ANTS. ANTS is a fluorescent molecule.
  9. For a fluorometer with computerized data acquisition, the assay can be calibrated by using the following equation:

where F(t) is the extent of fusion at time t, I (t) is the fluorescence intensity at time t, I(0) is the fluorescence intensity of the initial mixture of liposome encapsulated ANTS and liposome encapsulated DPX before adding the fusogen (step 3 and 4). I(∞) is the fluorescence intensity of the  liposome co-encapsulated ANTS/DPX (step 6).