This product includes 1.25 ml of liposomal dye, 1.25 ml of color development solution, 7.5 ml of buffer and 1 ml of DMSO. It is for assays of 100 samples using a 96-well plate.
Disruption of cell membranes that are composed of lipid bilayers leads to dramatic biological effects. For example, pore-forming cytotoxins disrupt the cell membrane permeability causing the release of inflammatory cytokines and tissue damage. The Lipid Bilayer Permeability Disruption Assay employs a liposome-encapsulated dye to monitor the release of the dye due to damage of the integrity of the lipid bilayer. Since liposomes have the bilayer structure similar to cell membranes, this dye-releasing assay may be used to test compounds that disrupt the permeability and damage cell membranes. The released dye is monitored by fluorescence at 580 nm (excitation at 540 nm). It is a high throughput assay using 96-well plates and is suitable for screening of compound collections.
The liposome bilayer is composed of lipids HSPC: cholesterol: DSPE-PEG2000 (3:1:1 mass ratio) which is the same as the lipids for an existing liposomal drug in clinic. The dye is rhodamine conjugated with diethylenetriamine and its fluorescence is quenched inside of the liposome. When the bilayer permeability is disrupted, the encapsulated dye is released and the fluorescence is generated in the presence of the color development agent outside of the liposome.
The Lipid Bilayer Permeability Disruption Assay Kit (Catalog number BPD100K) includes 1.25 ml of liposomal dye, 1.25 ml of color development solution, 7.5 ml of buffer and 1 ml of DMSO. It is for assays of 100 samples using a 96-well plate.
PROTOCOL
The following protocol is based on using a 96-well plate fluorescence plate reader (emission 580 nm, excitation 540 nm). The total sample volume is 100 µl. Cuvettes may also be used for fluorescence measurement if the sample volume is adjusted accordingly.
1. Temperature effect on bilayer permeability
Heating disrupts bilayer permeability and releases the liposome-encapsulated dye. To test the temperature effect, in each test tube, add 75 µl of Buffer, 12.5 µl of liposomal dye and 12.5 µl of color development solution. Incubate the tubes at different temperatures (e. g. boiling water bath, room temperature, etc) for 5 min. Transfer the samples into wells of a black 96-well plate and read the fluorescence at 580 nm (Excitation at 540 nm) in 10 min.
2. Control experiment of bilayer permeability disruption by DMSO
DMSO completely disrupts bilayer permeability and releases the liposome-encapsulated dye. In a well of a black 96-well plate, add 75 µl of DMSO, 12.5 µl of liposomal dye and 12.5 µl of color development solution. Incubate the sample at room temperature for 15 min and read the fluorescence at 580 nm (Excitation at 540 nm).
3. Disruption of bilayer permeability by compounds
Some compounds may also disrupt bilayer permeability and release the liposome-encapsulated dye. In a well of a black 96-well plate, add the compound solution and Buffer to make a volume of 75 µl then add 12.5 µl of liposomal dye. Incubate the tube at a proper temperature for a selected time period. Finally add 12.5 µl of color development solution and incubate the sample for 10 min. Read the fluorescence at 580 nm (Excitation at 540 nm).
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