This study investigates protein-phospholipid interactions on cell membranes using quantitative flow cytometry. The methodology involves the use of fluorescently-labeled proteins and phospholipid vesicles to analyze binding dynamics.
Protein-phospholipid interactions on the cell membrane are crucial for the regulation of several cellular processes.
To detect target protein-phospholipid interactions in vitro using quantitative flow cytometry, take a tube containing a desired concentration of cell membrane mimic — micron-sized, lipophilic dye-labeled phospholipid vesicles — in buffer.
Add an appropriate concentration of different fluorescently-labeled target proteins in solution.
Inject the suspension into the flow cytometer. Set the fluidics system parameters to a low flow rate to allow sufficient time for the protein-phospholipid interactions.
As the fluorescently-labeled proteins and phospholipid vesicles interact in the flow channel, the concentration of proteins bound to the vesicles increases.
As these complexes pass through the laser beams from specific fluorescence channels providing appropriate wavelengths, they become excited. These excited proteins and phospholipid vesicles in the complex emit respective fluorescence signals upon relaxation to the ground state, which are captured by the appropriate detectors.
The detection of a high mean fluorescence intensity of the proteins is indicative of protein binding to the phospholipid vesicles.
Start the kinetic binding experiments by diluting phospholipid vesicles in Tyrode's buffer to a concentration of 1 micromolar and a total volume of 250 microliters. Then, mix fluorescent-labeled coagulation factor X or fX-fd at a concentration of 500 nanomolar with the phospholipid vesicles in a 1:1 ratio to get a total volume of 500 microliters.
Immediately inject 500 microliters of the mixed suspension into the flow cytometer at a flow rate with an excitation wavelength of 488 nanometers and an emission filter of 585 nanometers with a width of 42 for channel FL2.
Next, measure the mean fluorescence in channel FL4 with excitation at 633 nanometers and emission filter at 660 nanometers with a width of 20. When the saturation of binding is achieved, rapidly dilute the sample 20-fold with Tyrode's buffer, and monitor the dissociation until a baseline fluorescence or a plateau is reached.
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