The study of the output of the immune synapse has been restricted mostly to microscopic methods. BSLBs have expanded our repertoire of tools that can be used to study the output of T cell immune synapses, including flow cytometry, microscopy, proteomics, and RNA sequencing technologies. As such, BSLBs can be used in a wide variety of immunology questions and in identifying the composition and biochemical regulation of the T cell output.
This includes, for instance, the study of chimeric antigen receptors, drugs that inhibit specific signaling pathways, or the genetic ablation of genes of interest. One of the most critical aspects of this protocol is to optimize the protein calibrations and the multicolor flow cytometry panel required for tracking the trans-synaptic particle of interest. This requires the systematic iteration of protein concentrations, antibody concentrations, and instrument settings.
Begin by diluting one microliter of bead solution with 1, 000 microliters of PBS. Count the beads using a hemocytometer chamber and calculate their concentration per milliliter. Then, calculate the volume of silica beads needed for 500, 000 final BSLBs per titration point.
Transfer the required volume of silica beads to a sterile 1.5-milliliter microcentrifuge tube. Wash the silica beads three times with one milliliter of sterile PBS and centrifuge the beads. Prepare three volumes of liposome master mix containing a final 12.5-mole percent of nickel-containing phospholipids.
Use the liposome master mix to resuspend the washed silica beads. Then, gently mix it by pipetting up and down half of the total volume. Using a sterile technique, add argon or nitrogen gas to the tube to displace air and protect the lipids from oxidation during mixing.
Add argon to the 0.4-millimolar lipid stocks. Store the stock and manipulate using a sterile technique. Move the BSLBs to the vertical mixer and mix for 30 minutes at room temperature using orbital mixing at 10 RPM.
Then, spin down the beads by centrifuging for 15 seconds at room temperature on a bench-top minicentrifuge. Block the formed BSLBs by adding one milliliter of 5%BSA containing 100-micromolar of nickel sulfate to saturate NTA sites on the BSLBs. Wash three times using HBS-HSA buffer to remove the excess blocking solution.
Take a new U-bottom or V-bottom 96-well plate and prepare two-fold serial dilutions of the proteins. Resuspend the prepared BSLBs in a volume such that 100 microliters of HBS-HSA buffer contain 500, 000 BSLBs. Then, take another U-bottom or V-bottom 96-well plate and transfer 100 microliters of the BSLB suspension to the wells in such a way that each well receives 500, 000 BSBLs.
Spin down the second plate at room temperature for two minutes at 300x G.Discard the supernatant and transfer 100-microliter volumes from the protein titration plate to the plate containing the sedimented BSLBs. Mix gently, avoiding excess bubble generation while pipetting. Use aluminum foil to protect it from light and incubate at room temperature for 30 minutes at 1, 000 RPM.
Then, wash the plate three times by adding HBS-HSA buffer to the plate and spin it down at room temperature for two minutes at 300x G.Count the cells after washing. Then, dilute them to a final concentration of 2.5 million per milliliter using synaptic transfer assay medium. Once the BSLBs are loaded with the protein mix, wash the BSLBs twice with HBS-HSA buffer to remove the excess unbound proteins.
Resuspend 500, 000 BSLBs per well in 200 microliters of HBS-HSA buffer. Transfer 100 microliters of BSLBs per well to a new U-bottom 96-well plate to make a duplicate, such that the final amount of BSLB per well is 250, 000. Spin down the BSLBs at 300x G for two minutes at room temperature.
Discard the supernatant and then resuspend the BSLBs using 100 microliters of T cell suspension. Mix gently to prevent the formation of bubbles. Incubate the co-cultures for 90 minutes at 37 degrees Celsius.
Protect the cells from light and cool down the co-cultures by first incubating them at room temperature for a minimum of 15 minutes. Centrifuge the co-cultures at room temperature for five minutes at 500x G.Discard the supernatant and resuspend the co-cultures in calcium and magnesium ion-free 2%BSA PBS at room temperature for blocking. Place the cells on ice for 45 minutes and protect them from light.
Prepare the antibody master mix using ice-cold 0.22-micrometer-filtered 2%BSA and PBS as a staining buffer. This master mix will provide extra blocking. Spin down the co-cultures at 500x G for five minutes and four degree Celsius.
Discard the supernatants, and then use a multi-channel pipette to resuspend the cells in the staining master mix containing optimized antibody concentrations. Include isotype-labeled cells and BSLBs, fluorescent and non-fluorescent BSLBs, and cells and BSLBs stained alone. Mix gently by pipetting up and down half of the volume.
Incubate for 30 minutes on ice and protect them from light. Wash the cells and BSLBs twice using ice-cold 2%BSA PBS. Spin them down at 500x G for five minutes at four degrees Celsius.
After centrifugation, check the co-culture sedimented on the bottom of the wells. Then, resuspend the washed co-cultures in 100 microliters of PBS and acquire immediately. Activate the log scale for side-scattered light and the time-of-flight parameter for side-and forward-scattered lights.
Acquire MESF standards, ensuring both the dim-set and brightest populations fall in the linear range of the instrument. Then, acquire compensation samples, calculate compensation, and apply the compensation matrix to the experiment. Acquire and save a minimum of 20, 000 total MESF standards for each quantification channel.
Avoid using FACS sheath fluid or similar to resuspend MESF beads, as these contain salts and alcohols that will destroy the fluorochromes, leading to wider fluorescence peaks and high error. For acquisition using high-throughput samplers, set instrument acquisition to Standard, set sample acquisition to 80 microliters. Sample Flow Rate between two and three microliters per second, sample Mixing Volume at 50 microliters, sample Mixing of 150 microliters per second, and mixing per well between three and five.
Acquire a minimum of 10, 000 single BSLBs per sample. Finally, export the FCS files. An example of quantitative flow cytometry measurements of ICAM-1 on the surface of tonsillar B cells and helper T-cells is shown here.
The representative images describe the gating strategy for analyzing single CXCR5 B cells and follicular-helper T cells isolated from human palatine tonsils. This sequential gating strategy identifies single live events within the continuous acquisition window. The doublets, the cell surface expression of ICAM-1 compared to FMO controls and FMO controls labeled with relevant isotypes of the populations, are shown here.
Gating and measurement of MFIs from different standard MESF populations in the overlaid histograms of the MESF populations are shown here. The values represent the MFIs for each of the five MESF populations. Linear regression of MESF over cMFI for the MESF populations are presented here.
The slope extracts MESF bound to cells from the FCM data of ICAM-1 on the surface of tonsillar B cells in TFH. The conversion to absolute molecular densities follows these simple mathematical operations. The representative images show the flow cytometry analysis of BSLBs reconstituted with increased densities of recombinant monomeric ICAM-1 12 histidines.
Regression analyses of ICAM-1 reference concentration over measured density are shown here. The slope is used to calculate target concentrations of protein to achieve the density of cells. The flow diagrams show the critical steps for co-culturing T cells with BSLBs, reconstituting model membranes in the subsequent measurement of particle transfer with flow cytometry.
The gating strategy to identify single BSLBs and cells within the continuous acquisition window is shown here. One of the most important aspects of this protocol is that, to maintain reproducibility of the results, each time you change protein or lipid stocks, you need to perform new calibration analysis. To discover new effective molecules secreted by different T cell subsets, BSLBs can be further isolated by fluorescence-activated cell sorting as subjected to transcriptomic and proteomic analysis.
Our colleagues within the University of Oxford have used this technology to study and demonstrate new receptor-ligand interactions, and also to study the effects of genetic ablations on the output of the T cell immune synapse.
