Encyclopedia of Experiments: Biological Techniques
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Transcript
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.
