Using this quantity flow cytometry method researchers can calculate the kinetic and equilibrium customs of brought in membrane interaction and estimate the number of protein brought in by sites on the PPH membrane. The advantages of this method are the simplicity and availability of reagents and equipment. This method is exclusively for basic research.
The protocol was developed to study protein lipid interactions in blood calculation, and can be used in other areas to characterize the interaction of proteins with various lipid membranes. The method can be used for the quantity assessment of ligand binding dynamics on the various cells and ligand types. Start the kinetic binding experiments by diluting phospholipid vesicles in Tyrode's buffer to a concentration of one micromolar and a total volume of 250 microliters.
Then mix fluorescent labeled coagulation factor X or FX FD and a concentration of 500 nanomolar with the phospholipid vesicles in a one to one 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 FL two.
Next measure the mean fluorescence in channel FL four 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. For equilibrium binding experiments incubate five micromolar artificial phospholipid vesicles with varying concentrations of FX FD from zero to 1000 nanomolar in Tyrode's buffer for 20 minutes.
After incubation dilute each sample by 20 fold to a final volume of 200 microliters with Tyrode's buffer then analyze the diluted sample by flow cytometry within 30 seconds. To analyze the data, export the experiments in FSC format from cytometry data acquisition software to cytometer software for data analysis. By choosing the file and then clicking the export and FSC files tabs.
once done open the FCS files into cytometer software by selecting the files on the computer and dragging the files to the program's workspace. For gating the microvesicles, identify the region of microvesicles by fluorescence of the lipophilic dye, DIC 16 three. Then use the plot button in the worksheet to create a dot plot SSC from FL two D I C 16 in log coordinates.
Then choose the rectangular gate button to draw a gating region. To analyze the kinetic experiments create a dot plot using the coordinates of fluorescence over the time for the region of the vesicles. Next, export the data on the change in fluorescence over time in the CSV format;by going to choose sample and right clicking on the export tab, followed by choosing FL four time in parameters, select directory for saving before clicking the select CSV format and hitting the export tab.
After transfer, open the CSV file in statistical software to calculate the simple moving average fluorescence and time for every 1000 events approximate a graph of the dependence of the single moving average fluorescence on time under the assumption of exponential dependence and use the value to calculate the kinetic association constant using an equation. Then calculate the kinetic dissociation constant using the equation as described earlier. To analyze the date of the equilibrium binding assay, determine the average fluorescence of FX FD in the region of the vesicles for each selected concentration of FX FD and then approximate the dependence of the bound factor fluorescence from the concentration of the added factor in the assumption of single site binding.
Calculate the average binding parameters using an equation from three independent repeats at a minimum. Proceed to prepare calibrated beads by incubating gel filtered platelets with calcium IANA 4A twenty three, one hundred and eighty seven in the presence of 2.5 millimolar calcium chloride for 10 minutes at room temperature. Do the activated platelets add the various concentrations of FX FD and incubate the platelets with two volume percent formaldehyde.
After one hour, stop the reaction by incubating the platelets with three molar glycine and 5%BSA for 30 minutes at room temperature. Then purify the mixture from the unreacted dye with centrifugation at 400 times G for five minutes. Remove the supernate before resuspending the pellet in Tyrode's buffer containing 0.5%BSA.
Next, measure the fluorescence level of the calibration beads in each sample using a spectrofluorometer and then using the flow cytometer. Determine the number of beads in each sample with the help of a cell counter. Convert the fluorescence intensity of each respective bead sample to the concentration of soluble fluorescent dye using a spectrofluorometer and recalculate the fluorescent dye concentration for the number of spectrofluorometer molecules.
Create a dependence graph of the average fluorescence of the beads in a flow cytometer on the number of flurophore molecules for each sample. Then approximate the dependence by line proportionality using the analysis fitting and fit linear tabs in order. From the approximation in the equation, calculate the conversion factor of the mean fluorescence to binding sites.
Later calculate the apparent number of the binding sites per vesicle of interest. The representative analysis shows gating of the artificial phosphor lipid vesicles that are one micrometer in size with the incorporated lipophilic fluorescent dye, dye I C 16, the gate was set based on a sample containing the same size artificial lipid vesicles without the fluorescent dye. The kinetics of the protein binding to the vesicles was analyzed at the first stage by collecting the sample continuously.
The data obtained were analyzed using flow cytometry. The flow cytometric measurements should start as soon as possible after mixing the solutions and diluting with Tyrode's buffer. The proposed method could be supplemented with surface plasma resonance for studying brought in membrane interactions which is highly sensitive with good temporary resolution and doesn't require brought in delivery The main advantage of this method is its accessibility and simplicity.
