Fluorescência Biomembrane Força Probe: quantificação simultânea de Kinetics receptor de ligando e induzida Encadernação-Sinalização Intracelular em uma única célula

The overall goal of this procedure is to simultaneously measure receptor like and binding and to observe the binding induced calcium signaling on a single cell. This is accomplished by first isolating biotin and adjusting the osmolarity of human red blood cells or RBCs to use them as an ultra-sensitive force transducer. The second step is to functionalize microscale glass beads, which includes cleaning, dilation and ligand coating.

Next, the T cells are isolated and incubated with the calcium dye fewer 2:00 AM, which enables the observation of intracellular calcium ion concentration. The final step is to prepare the micro pipettes in experimental chamber that are specially required for the experiment setup. Ultimately, the fluorescence bio membrane force probe or BFP technique is used to show the adhesion of a cell to a ligand coated bead, measure the receptor ligand binding kinetics, and meanwhile monitor the intracellular calcium level.

This technique is useful in understanding how mechanical force is sensed by T cells via androgen recognition. A major advantage of this technique over existing methods is that FBIP can measure receptor ligand binding kinetics and an intracellular signaling simultaneously. The challenging parts of this protocol include manually producing micro pep paths of the correct diameters, picking up cells and bees with micro PEPs and assembling the force probe, which can fail at first, Although he could not be here for the demonstration of this video.

Co-author Arnold G, who has recently completed his PhD in my laboratory, has contributed significantly to the initial fabrication validation of the FBAP protocol. Obtain eight to 10 microliters of blood from a finger prick and add to one milliliter of carbonate bicarbonate, buffer jet leave vortex or pipette the mixture and centrifuge for one minute at 900 G.Discard the supernatant before performing the carbonate bicarbonate buffer. Wash once more.

Next, mix 10 microliters of the resulting RBC pack 171 microliters of carbonate bicarbonate buffer, and 1049 microliters of biotin. PEG NHS linker solution and incubate at room temperature for 30 minutes. Using the same washing procedures before wash the resulting RBC mixture once with carbonate bicarbonate buffer, and then twice with N two 5%buffer.

Next dilute Nystatin into N two 5%buffer. To make a final concentration of 40 microliters per milliliter, mix five microliters of the biotinylated RBC with 71.4 microliters of Nystatin solution and incubate for one hour at zero degrees Celsius. Finally, wash the samples twice with N two 5%buffer and store the biotinylated RBC with N two 5%buffer plus 0.5%BSA in the refrigerator mount one piece of three inch micro pipette onto the micro pipette puller.

Click the pull button so that the middle of the capillary will be heated by the machine and the capillary will be pulled on the two ends to make two capillaries with sharp tips, which are the raw pipettes. Next mount a raw pipette onto the pipette holder of the micro pipette Forge heat to melt the glass sphere on the forge. Insert the tip of the raw pipette inside the glass sphere.

Cool down the glass sphere and pull the raw pipette to break it from the outside, leaving its tip inside the sphere. Repeat this procedure until the desired tip orifice is obtained to build the cell chamber first cut to 40 millimeter by 22 millimeter by 0.2 millimeter cover slip using a glass cutter into two 40 millimeter by 11 millimeter by 0.2 millimeter pieces. The cell chamber is built on the foundation of a homemade chamber holder consisting of two pieces of metal squares and a handle that links them together.

Using grace glue one cover slip to the bottom side of the chamber holder in a way that it bridges the two metal squares. Similarly, glue the other cover slip to the top side, which will form a parallel cover slip cell chamber. Next, use the pipette to inject 200 microliters of experimental buffer in between the two cover slips.

Make sure the buffer attaches to both cover slips. Gently rotate and shake the chamber to let the buffer touch both ends of the chamber. Then carefully inject mineral oil into both sides of the chamber, flanking the experimental buffer zone, thereby sealing the buffer from the open air.

Inject suspensions of prob. Beads are bcs and targets in the upper, middle, and lower regions of the buffer zone respectively. To begin the BFP experiment, turn on the microscope and light source.

Place the chamber onto the main microscope stage. The next step is to install all three micro pipettes of the BFP. The probe to the left is to grab A RBC.

The target to the right is to grab the cell and the helper to the lower right serves to grab a bead to install, use a micro injector to backfill a micro pipette with experimental buffer. Take the micro pipette off the pipette holder and hold it at a lower place to allow liquid to drip from the tip. Quickly insert the micro pipette into the holder tip, making sure no air bubble gets into the micro pipette.

During the insertion, tighten the holder screw, then mount each pipette holder onto its corresponding micro manipulator. Push the micro pipette towards the chamber so that their tips enter the chamber buffer area. Adjust a position of the micro pipette and find them under the microscope field of view.

Move around the chamber holder stage to find the colonies of the three elements. One by one, adjust the position of the corresponding micro pipette by turning the knobs of the manipulators to let the tip of the micro pipette approach one cell or bead, aspirate the cell or bead. By adjusting the pressure inside the corresponding micro pipette, all three micro pipettes will capture their corresponding elements.

Move around the chamber holder stage to find an open space away from the colonies of the injected elements where the experiment will be performed. Switch the microscope visual mode to visualize the image on the computer program. Move all three elements on the pipette tips into the program’s vision field.

Next, align the probe bead and the RBC and carefully maneuver the probe bead to the apex of the RBC. Now briefly impinge the bead onto the RBC and gently retract, adjust the pressure of the helper micropipet to gently blow the bead away so that it would be left glued onto the RBC apex. Move the helper pipette away and align the target and prob bead on the program.

In the vision field window, use the program tools to measure the respective radii of the probe micro pipette, the RBC and the circular contact area between the RBC and the probe bead. These values allow for the estimation of the spring constant of the RBC by the equation listed in the text protocol. After entering the desired RBC spring constant into the program, draw a horizontal line across the RBC apex, which will yield a curve in the adjacent window indicating the brightness of each pixel.

Along this line, drag the threshold line to be at around half the depth of the curve. Next, select the desired experiment mode, thermal fluctuation assay adhesion frequency assay, or force clamp assay. Set the parameters as desired.

Click start, which allows the program to move the target pipette and drive the target in and out of contact with the probe data collection will be performed and parallel, which records the position of the pro bead in real time. Stop the program by clicking on the button stop experiment at which a time a window will pop out. To allow saving the acquired data to use the fluorescence function of the BFP system turned on the excitation light source and the fluorescence camera, which are controlled by a separate program.

Then select the parameters for the fluorescence imaging, including gain exposure and excitation channels. Follow all preparations in the BFP experiment protocol, including aligning the probe and the target to allow for visualization of the target cell live fluorescent image that is excited by 340 nanometer or 380 nanometer excitation light. Use the sectioning tool to roughly section the area within which the cell will stay during the entire recording period.

Next, simultaneously click on record and start clicking. Record allows a 340 nanometer and 380 nanometer light to alternately excite the intracellular fluorescence die. A pair of corresponding fluorescence images will be alternately recorded about one every second.

Clicking on start begins the BFP experiment for analysis of molecular interaction and the fluorescence imaging experiment to monitor intracellular calcium signaling proceed to analyze the data as described in the text protocol. In time force raw data curves, the force is derived from tracking the position change of the pro bead. In a no adhesion event, the force will return to zero in the force reverse time signal after the target cell is retracted from the bead surface.

Conversely, in a rupture force event, the adhesion will be manifested by a quick elevation of force to a positive level during retraction, followed by a quick drop back to zero. In a lifetime event, the positive force will be clamped to realize a plateau. The length of the plateau is a lifetime.

By repeated conducting VFP experiment cycles, a force versus time curve will be derived. Shown here are the fluorescent images of a few or two loaded T-cell upon excitation by 340 nanometer and 380 nanometer light channels. Both images show clear contours of the cell, indicating the cell was stained well After the binding kinetics data and fluorescence data were derived from a T cell, both the cumulative lifetime curve and the calcium level curve are presented in the same figure to find the correlation between them.

Now you should have a good understanding of how to perform A-F-B-I-P experiment. During this procedure. It is important to remember to check if there’s any defect in the force probe, like the diameter of the micro pipet, the length of the rubber cell projection, and the position of the bead.

All this will affect the accuracy of the force measurement. This technique paves the way for researchers in the field of molecular mechanical biology to study the mechanism of mechanical transduction in all possible biological systems.