This method makes it possible to measure the spatial temporal distribution of the forces applied by B cells at immune synapse and correlate them with the recruitment of specific proteins. Traction force microscopy using polyacrylamide gels is easy to implement. This protocol can be used to quickly set up the measurement of the mechanical capabilities of many B cells.
This method is flexible and can be adapted to graph other ligands like integrins or to study other kinds of immune synapses like T cells or frustrated phagocytosis. Due to the physical and chemical properties of the gels required by this experiment, the adaptation of classical traction force microscopy methods to B cells may be tricky. Begin by salinizing the gel support.
Activate the coverslip or glass bottom Petri dish with a UV lamp for two minutes, then salinize it with 200 microliters of APTMS for five minutes. This will prepare the support for the covalent binding of the gel. Thoroughly wash the coverslip or glass bottom dish with ultra pure water and dry it using vacuum aspiration.
To prepare the coverslips for flattening the gel, put them into a ceramic coverslip holder, put the holder in a small beaker, and pour siliconizing reagent over the coverslips making sure to cover them completely. Cover the beaker with aluminum foil and leave it at room temperature for three minutes. Meanwhile, fill a large beaker with ultra pure water.
After three minutes of incubation in siliconizing reagent, transfer the coverslip holder with the coverslips to the beaker with water. Thoroughly rinse the coverslips with ultra pure water. Dry them well and place them on paper wipes.
For best results, immediately proceed with gel polymerization. Prepare a 500 Pascal gel pre-mix according to manuscript directions, then combine 167 microliters of the pre-mix with 1.67 microliters of beads. Vortex and sonicate the mixture in a bath sonicator for five minutes.
Protect the mix from light with aluminum foil. To capitalize polymerization, add 1.67 microliters of 10%ammonium persulfate to the gel mix, then initiate polymerization by adding 0.2 microliters of TEMED and mixing the gel with a pipette. To cast the gel, pipette nine microliters of gel mix onto each salinized coverslip or glass bottom dish.
Immediately flatten the gel with the siliconized coverslip, pressing down on it with forceps to ensure that the gel spreads across the entire area of the coverslip and that some of it leaks out. Invert the coverslip or glass bottom dish into a large Petri dish and tap it on the bench to force the beads towards the gel surface. Place a humidified tissue on the dish to create a wet chamber.
Cover it with aluminum foil and incubate it for one hour. After the incubation, facilitate coverslip release by adding PBS to the sample. Carefully remove the coverslip with a needle, slightly tilting the dish but making sure that the gel is submerged in the PBS.
The siliconizing agent, acrylamide, base acrylamide, and TEMED can be toxic by inhalation. Wear standard personal protective equipment and manipulate these products under a chemical hood. Aspirate the PBS from the gels and add 150 microliters of Sulfo-SANPAH at room temperature.
Expose the gel to UV treatment for two minutes, then wash the gel three times with PBS. Repeat the treatment with Sulfo-SANPAH and the PBS washes, then add 250 microliters of hen egg lysozyme or HEL to the gel and incubate it overnight in a humidity chamber at four degrees Celsius covered with aluminum foil. After the incubation, remove the HEL antigen and wash the gel with PBS three times.
Finally, cover the gel with 500 microliters of B cell culture media and leave it at room temperature. Use a confocal microscope with thermal and carbon dioxide control for imaging. Aspirate the media from the gel, leaving approximately 200 microliters.
Position the gel on the microscope. Two main layers of beads will appear on the bottom and top of the gel. Focus on the gel plane and find an even area to image.
Pick the imaging area carefully and make sure to stay in focus. An appropriate bead density and an even surface are key to obtaining robust and reliable force measurements. Add 80 microliters of primary B lymphocytes from MD4 mice to the gel, avoiding touching the gel to maintain focus.
Ensure that the focus is still correct and that cells can be seen descending in the area. Launch the acquisition before the cells reach the gel. Open the movie as a stack of images in ImageJ, then run the macro cropandsave.ijm.
Choose an output directory and configure the attribute channel settings. Select the regions of interest with the rectangle tool and add them to the ROI list using the T key. When finished, click OK.When the macro proposes a mask of the cell, click OK if it is satisfactory.
If not satisfactory, click not OK and then manually select a closed region with any selection tool, then click continue. Open MATLAB and run tfmv1.m. Input the required parameters.
Specifically, check image properties, such as pixel size and time interval of acquisition and the gel properties such as Young modulus E and Poisson ratio. When finished, locate the outputs of the software in the same directory as the original file. Correct bead images look like a uniform and random distribution of bright spots similar to a starry sky.
Data and analysis are not reliable when the number of beads is too low or the image is out of focus. It is possible to observe the movement of beads by eye using a reference frame that preceded the first contact of the cell with the substrate. Approximate results could be obtained from single particle tracking.
The analysis provides a segmentation of the beads in the reference image as a control. Using software, it is also possible to obtain the displacement and stress field, which is the vector of the local stress at each pixel and each time point. Scalar product of the displacement and force fields integrated on the area of the cell provides total work exerted by the cell on the substrate.
When comparing two biological conditions, an average curve or an average value over the last time points where the energy reaches a plateau can be calculated. When spatial information of the forces is relevant, it is also possible to compare single time points of each condition. An example of fluorescence antigen extraction time-lapse is shown here.
The progressive appearance of fluorescent signals at the synapse indicates antigen detachment from the gel. The fluorescence data can be used to construct an average extraction curve. The most delicate step in the protocol is the polymerization of the gel under the coverslip.
This step must be performed relatively quickly and carefully ensuring that the gel is squeezed uniformly under the coverslip. Following this procedure, fluorescent protein expressing B lymphocytes can be used to simultaneously assess the localization of intracellular structures and force patterning. This technique can be combined with genetic or chemical perturbations to assess the role of specific proteins on cell contractility and antigen uptake.
