The overall goal of the following experiment is to measure receptor ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells. This is achieved by first functionalizing, the red blood cells, which are the adhesion sensors with ligands and preparing receptor bearing cells. As a second step, the micro pipette chamber is assembled and adhesion tests are performed to record the adhesion events.
Next, the adhesion probability versus cells contact time curve is plotted and fitted with the model. In order to estimate the kinetics parameters, results are obtained that show two dimensional effective binding affinity and off rate estimated from the data. So where do we get the idea of this assay?
People study receptor leg interaction. Mostly measure ruptured forces and we are doing the same thing, but one day we thought could we just measure the binary outcome zero for no adhesion and one for adhesion and derive the measurement for kinetics from those zero one measurements to show the micro pipee frequency assay. Dr.Veronica Zana Zina, a research scientist of my laboratory, will show the experiment to you.
Red blood cells isolated from whole blood are biotinylated for the adhesion frequency assay. To begin this procedure, centrifuge the tube containing isolated red blood cells, aliquot 10 microliters of red blood cell pellet into each of several vials and add the corresponding amount of PBS to each vial to prepare multiple red blood cell biotin elation concentrations. Next, add 10 microliters of 0.1 molar bore eight buffer to each vial.
Then add a calculated amount of biotin solution to each vial and vortex immediately. For several seconds, place each red blood cell vial inside a 50 milliliter conical tube and incubate on a rotator for 30 minutes at room temperature after 30 minutes, add EAS 45 buffer to each vial and centrifuge for two minutes at 2000 rotations per minute. Repeat the wash with EAS 45 buffer twice for a total of three washes.
Finally, add 100 microliters of EAS 45 buffer to each vial and store at four degrees Celsius to functionalize the ligands on the red blood cell surface by at inated in the previous step, red blood cells are incubated with a saturated concentration of strep TTR in for 30 minutes at four degrees Celsius and wash three times with EAS 45 buffer for two minutes at 2000 rotations per minute. After that, either incubate the red blood cells directly with the biotinylated ligand or incubate the red blood cells with the corresponding biotinylated capturing antibody followed by incubation with ligands. The protocol shown in this video uses the biotinylated capturing antibody.
Micropipets are made using ax melting point bo silicate glass capillary tubes. First use a micro pipette puller to pull the capillary tubes. Next, use a micro manipulator with a micro forge to cut the tip of the micro pipette to the desired size opening.
The required diameter usually ranges from one to five microns depending on the size of the cells to be used in the study. To prepare the cell chamber, use two pieces of microscope cover glass cut to the desired size, clean the dust from the cover slips with an air duster, add the cell chamber medium and seal the chamber using mineral oil from both sides to avoid medium evaporation. That will change the osmolarity during the experiment.
The micro pipette system used in this assay consists of three subsystems, an imaging subsystem for observing, recording and analyzing movements of the micro pipet aspirated cell, a micro-manipulation subsystem to enable the selection of cells from the cell chamber and a pressure subsystem for aspirating the cells into micro pipettes. The central piece of the imaging subsystem is an inverted microscope with a 100 times oil immersion. Objective images are detected by a charge couple device camera and sent to a video cassette recorder.
A video timer is coupled to the system to keep track of time. After filling the micro pipette with deionized water, place it in its holder on a mechanical drive mounted on the microscope. The micro pipette can be finely positioned with a three axis hydraulic microm manipulator.
One of the micro pipette holders is mounted on a computer programmed piezoelectric translator. This allows the pipette to be moved precisely and in an adhesion test cycle. The pressure regulation subsystem is used to control suction.
During the experiment, a hydraulic line connects the micropipet holder to a fluid reservoir. A fine mechanical positioner allows the height of the reservoir to be precisely manipulated. After the chamber is assembled, a micro pipettes are set, inject the cells into the chamber to begin the assay, aspirate the interacting cells by the respective pipettes and use the PSO electric translator to drive the red blood cell in and outta contact with the neutrophil with controlled contact area and time.
An adian event is observed when the red blood cell elongates upon cell separation, a no adhesion event corresponds to no red blood cell elongation. Record the observed adhesion events by adding one for adhesion or zero for no adhesion. In a column of an Excel spreadsheet.
Record the adhesion frequency versus contact time curve using at least three different cell pairs for each contact time. To obtain a mean and SEM as a control record the non-specific binding curve by using red blood cells coated with a relevant ligands and or blocking the ligands or receptors using their specific functional blockade. Monoclonal antibodies, specific adhesion frequency at each contact time point can be calculated by removal of the non-specific adhesion frequency.
The specific adhesion frequency versus contact time data is fitted by a probabilistic model that describes a second order forward and first order. Reverse single step interaction between a single species of receptors and a single species of ligands in this equation, KA is the 2D affinity. KA is the 2D off rate.
MR and ML are the respective receptor and ligand densities and AC is the contact area. The curve fit has two parameters, ac, k, a, and K off as AC and KA are lumped together and called collectively as effective 2D affinity its product with the off rate is the effective 2D on rate AC K on equals AC KA times K off. Shown here is an example of the determination of integrin alpha L beta two.
Site density on neutrophils. Neutrophils were first incubated, one microgram per milliliter of E selectin IG and then were saturating concentrations of PE conjugated anti-human CD 11, a monoclonal antibody or a relevant mouse IgG one as a control. After 30 minutes incubation samples were washed and read on a flow cytometer with standard quantity.
Bright PE calibration beads. Panel A shows fluorescence histograms of calibration beads together with those of E selectin IG treated or untreated cells. Specific CD 11 A monoclonal antibody staining is shown in solid curves and relevant isotype match control antibody staining is shown in dotted curves.
Panel B shows the process of density quantification. Log 10 was calculated for the mean fluorescent intensity of each of four beads. Calibration peak value from panel A and for the lot specific PE molecules per bead.
A linear regression of log 10 PE molecules per bead against log 10 fluorescence was plotted for reselect intruded cells. Log 10 fi is equal to 3.99 indicated with the blue solid circle and 2.23 indicated with the blue open circle for the specific monoclonal antibody and to control antibody respectively. When the linear equation for X was solved, the total number of alpha L beta two or neutrophils was calculated as 9, 587.
Surface density was calculated to be 43 sites per micron squared using 8.4 microns as the neutrophil diameter. The next graph shows the running adhesion frequency fi for specific binding at one second and 10 seconds. Contact times measured from repeated adhesion test cycles between ICAM one coated red blood cells and human neutrophils expressing integrin alpha L beta two.
In contrast shown here is the running adhesion frequency fi for nonspecific binding at one second and ten second contact times measured from repeated adion test cycles between human IgG coated red blood cells and human neutrophils expressing integrin alpha L beta two. This final graph illustrates the kinetics of I A one binding to neutrophil integrin alpha L beta two adhesion probabilities measured for three cell pairs at each contact time were averaged and plotted versus contact time to control for non-specific binding. Two different conditions were used.
One red blood cells coated with the anti TFC capture antibody and incubated with human IgG instead of ICAM one IG and two neutrophils binding to red blood cells not coated with the capture antibody. Non-specific binding recorded as human IgG control curve was used to obtain a specific adhesion probability curve. Using previously shown equation for non-specific binding correction.
The corrected specific adhesion probability curve was fitted with the model fitting. Specific adhesion probability curve returned effective binding affinity. A CKA equals 1.4 times 10 to the minus four microns to the fourth power and koff equals 0.3 s minus one.
After watching this video, you should have a good understanding of how to measure receptor ligand interaction kinetics when both molecule anchors on the surfaces of the I interaction cells. While attempting this procedure, it's important to remember the first workouts, ligand side densities range so that the adhesion of frequency would be in the mid range and remember that working with blood and some of the regions for specific treatment, for example, sodium that and DM so can be extremely hazardous. Therefore, precautions such as careful reading of the reagents MSDS and wearing gloves while performing this procedure are required.
