A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

This video describes a procedure for tracking T-cell receptor signaling events using dual channel simultaneous acquisition. Total internal reflection fluorescence microscopy or turf microscopy first T-cell are transfected with a plasmid encoding GFP tagged ZAP 70 a glass supported lipid bilayer, which incorporates lipid anchored peptide. MHC complexes, adhesion molecules, and co-stimulatory molecules is prepared.

The ZAP 70 transfected T cells are added to the bilayer when the T-cell receptors are engaged by peptide MHC complexes. The T-cell receptor associated CD three chains are phosphorylated by the SARC family kinase LCK, and the kinase zap 70 is recruited from the cytoplasm. The T-cell receptor is then fluorescently labeled and imaging is performed in which the cells are immersed in medium with lower refractive index than the cover glass and illuminated by a laser beam at an angle that undergoes total internal reflection.

This generates an evanescent wave at the glass water interface, which decays exponentially into the medium, allowing a penetration depth of 100 to 200 nanometers. Thus only fluoro fours in the plasma membrane or very close to it are excited. Analysis of the resulting images reveals signaling takes place in T cell receptor micro clusters.

The main advantage of turf microscopy or conventional confocal microscopy is that you get better resolution such that signaling clusters or signaling complexes such as TCR micro clusters are easily visualized. Travis Crites and Chen. Both postdocs in my lab will demonstrate how to transfect primary mouse T cells using the maxi nucleo affection technology and then image them using total internal reflection fluorescence microscopy or turf M stimulated with glass of bird lipid bile layer containing antigen.

This method has been extensively used to study signaling in various immune cells including T cells, B cells, natural killer, and master cells. The method is broadly applicable and can be used to study most receptor systems in mammalian cells, especially if signaling occurs in clusters. Individuals new to the method may struggle initially as T-cells are very hard to transfect.

In addition, a precise coordination is required so that T-cell viability and transfection efficiency, the bilar quality as well as microscope alignment are up to the mark. The first step of this procedure is to transfect either naive or in vitro activated primary T-cells with expression plasmids and coding signaling molecules tagged by GFP using an AM maxa mouse T-cell nucleo effector kit. All of the T cells used in this study expressed the A-N-D-T-C-R that recognizes MCC peptide found to the MHC molecule IE of K.The transfection procedure shown here is carried out according to manufacturer's recommendations.

However, some tips are presented that promote viability of the T cells after transfection. Both viability and transfection efficiency are higher in in vitro activated T cells than naive cells and hence, this protocol uses in vitro activated T cells before starting. Prepare two milliliters of supplemented T-cell medium for each transfection by combining 1.9 milliliters of medium provided in the kit 20 microliters component, A 20 microliters component B, and 100 microliters of fetal bovine serum in a two milliliter tube.

Transfer two milliliters of medium to each well of a 12 well plate. Place the plate in a humidified incubator at 37 degrees Celsius for at least an hour to allow the medium to equilibrate cell death will result from Resus suspension of transfected cells in medium that is cold or is not at a pH of 7.2. To prepare the transfection mixture, combine 82 microliters of mouse T-cell nucleo effector solution with 18 microliters of supplement two and five micrograms of endotoxin free plasmid DNA at a concentration no lower than 0.5 micrograms per microliter mixed thoroughly by tapping.

Transfer five to 10 million cells to a 15 milliliter centrifuge tube, centrifuge at 75 times G for five minutes at room temperature following the spin. Use a vacuum aspirator to remove as much of the supernatant as possible to resuspend the cell pellet in the transfection mixture gently and slowly aspirate the cell pellet three to four times until there are no large clumps of cells remaining. Using a pipette carefully transferred the suspension into a certified amaxa vete, ensuring that there are no bubbles present.

Cap the qve select nuclear effector program X 0 0 1 on the nucleo effector device. Insert the vete into the Q vete holder and press the button to apply the selected program. Then immediately transfer the vete and pre equilibrated culture medium to the hood.

Using the provided plastic pipette, add approximately 500 microliters of the pre equilibrated medium to the vete. Then add the cell suspension to the medium in the plate. Drop by drop.

Incubate the cells at 37 degrees Celsius for four hours to allow low level expression of the GFP tagged proteins and to minimize the appearance of artifacts from overexpression. In this section, a method for aligning the microscope for two channel simultaneous imaging is described before starting power on all components of the microscope. Start the computer and software.

Then ensure that all hardware components are recognized by the software. Since the software starts without any error messages, all components have been detected. Dilute one microliter of sub resolution.

The Flores bright multi fluorescent microspheres into two milliliters of PBS for use as a colocalization standard. Then add 0.25 milliliters of the microsphere suspension to each well of a lab tech eight well chambered cover glass system. Place the chamber slide system onto the platform above the objective.

Then bring the beads absorb to the glass into focus on the live preview. The left pane is the channel in which GFP transfected cells will be visible. The right pane will show labeled TCR during live cell imaging experiments.

Using the Y alignment of the XY, Z fiber launch, adjust the position of the laser beam such that the beam emerges out of the objective and is projected onto the ceiling. It is important to do this when the objective is in the position where the glass plane is in focus. If the beam on the ceiling is diffused, the beam is not collated.

Adjust the Z micrometer to achieve full collation test collation for each of the laser lines to be used in the experiment. Then using a laser power meter, adjust the laser power to 20 to 30 micro watts for each channel. T cells are extremely sensitive to laser radiation and the illumination is limited to a combined total of 50 micro watts.

Next to set the angle for turf. Turn the Y alignment micrometer on the fiber launch to move the beam away from the ceiling until its angle. With respect to the objectives, optical axis approaches 90 degrees.

Once the critical angle for total internal reflection is reached, the laser light will no longer exit the objective To assess whether turf alignment is achieved, focus up and down through the plane of the cover glass. Notice here that as the beads absorb to the surface are brought out of focus, many beads can be seen floating in solution. To achieve good turf alignment, it is necessary to increase the angle of the beam with respect to the objective.

As the angle of the beam is increased, beads that are floating near the surface begin to disappear and the beads absorbed at the interface increase in intensity. Now, when the beads add absorbed to the interface are brought out of focus, no beads floating in solution remain visible. At this point, the turf is properly aligned as only the beads that are absorbed to the cover slip are able to be in.Focus.

Next, to align the red and green fluorescent channels with respect to one another, focus on the beads and compare the position of identifiable features in the images produced in both channels. Using the X and Y micrometer screws on one camera stand, bring the relative position of the beads into as close proximity as possible.Here. Notice the bright bead in the lower left quadrant of both images to ensure spatial alignment between the two channels.

This feature is brought to the same relative position in both images. Capture and save the image from both channels. These images will be used as a colocalization standard to align the two channels.

An example of this alignment is seen here prior to alignment. One of the channels is rotated with respect to the other as shown in this image in which images from the two channels of the sub resolution beads are superimposed. Following adjustments, the images are aligned as shown here.

The quality of the alignment may be further scrutinized by comparing images of GFP Transfected living cells. Notice how the Laila podium from the two channels are perfectly aligned. After aligning the two images using the calibration established using the fluorescent microbeads.

Once the microscope is set up flow chambers containing glass supported lipid bilayers presenting antigen are prepared and turf M is performed to visualize TRANSFECTED T cells interacting with the substrate first. Following the protocol described by Ana and Dustin assemble a flow chamber with planar lipid bilayers formed on a glass cover slip containing NI NTA lipids, the adhesion molecule ICAM one and peptide MHC complexes. MCC loaded IEFK are incorporated in the bilayer via histidine tags.

The co-stimulatory molecule CD 80 is incorporated. Using A GPI anchor, place the flow cell on the stage of the microscope and stabilize it by attaching the chamber to an adapter plate on the microscope stage using a tension bar to immobilize it. Next, attach the manufacturer supplied heating element on the flow cell to control its temperature.

Position the objective on a bilayer and bring the bilayer plane into focus to enable heating of the objective and the flow cell switch on the flow cell and objective heaters to bring the temperature of the chamber to 37 degrees Celsius. After four hours of incubation, transfer the transected T cells into a 15 milliliter tube and centrifuge at a speed of 80 times G.Resuspend the cells in 300 microliters of H-B-S-B-S-A buffer. Then add H 57 FAB or H 57 single chain variable fragment to label the TCR to a final concentration of 10 micrograms per milliliter for fab or 20 micrograms per milliliter for the single chain variable fragment.

Transfer the cells to a five milliliter fax tube. Next, using a one milliliter slip tip syringe, inject the cells into the flow chamber via a three-way valve. Set up the imaging conditions so that the dual channel live acquisition shows a live preview of the TCR and GFP channels and find a field with transfected cells.

Due to intensity differences between the TCR and GFP, the scaling of the images may have to be adjusted in real time so that cells can be visualized in both channels. As is the case here, confirm turf illumination by focusing up the glass plane to detect any lateral membrane staining. If lateral membranes do not come in focus, then the turf alignment is good.

In this live preview lateral membrane staining is apparent both in the plane of the cover slip and when the cover slip is brought out of focus to bring the microscope into good turf alignment. The angle of the laser with respect to the objective is increased and the lateral membrane goes out of focus. Begin capturing individual images and time-lapse sequences with an interval of 10 seconds.

For five minutes, the cells were incubated on glass supported and INTA bilayers containing six molecules per square. Micrometer of peptide loaded hist tagged IEFK 100 molecules per square micrometer of Alexa 6 4 7 conjugated hist tagged ICAM one and 100 molecules per square. Micrometer of GPI anchored CD 80 dual channel Simultaneous acquisition turf microscopy was performed in the continuous presence of Alexa 5 46 conjugated H five seven FAB fragment.

To stain the TCR cells were imaged up to one hour after initial contact with the bilayer. Numbers of ZAP 70 clusters per cell were analyzed using an automated cluster counting software. At least 40 cells were analyzed in each case.

The peptides loaded are indicated in the inset. ICAM one refers to cells on bilayers containing 100 molecules per square micrometer of Alexa 6 4 7 conjugated. His tagged ICAM one as shown in the figure zap 70 dependent signaling occurs in TCR micro clusters and only in response to agonist ligands as can be seen in this movie, taken at 10 seconds per frame.

TCR signaling in response to agonist peptides is sustained by the continuous generation of TCR micro clusters in the periphery of the contact area that recruit ZAP 70. After watching this video, you should have a good understanding of how to transfect primary T-cells using a maxa nuclear affection technology and properly image the recruitment of GFPT signaling molecules to TCR micro clusters using turf microscopy. Once master, this technique can be done in seven hours if it is performed properly.

While attempting this procedure, it is important to remember to be very gentle with your primary cells. The healthy the cells are at each step of the experiment, the higher your transfection efficiency will be and the more cells you will capture in your imaging. T cells are highly mt and as a result, sequential imaging of TCR and GFP channel causes the two images to not align perfectly due to the movement of TCR micro clusters and cells.

To overcome this problem, we developed the dual channel simultaneous acquisition turf microscope.