We are grateful to our laboratory members and the Kennedy Institute of Rheumatology community for constructive scientific discussions, especially our flow cytometry facility manager Jonathan Webber. This work was funded by Wellcome Trust Principal Research Fellowship 100262Z/12/Z, the ERC Advanced Grant (SYNECT AdG 670930), and the Kennedy Trust for Rheumatology Research (KTRR) (all three to MLD). PFCD was supported by EMBO Long-Term Fellowship (ALTF 1420-2015, in conjunction with the European Commission (LTFCOFUND2013, GA-2013-609409) and Marie Sklodowska-Curie Actions) and an Oxford-Bristol Myers Squibb Fellowship.

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The authors have no conflict of interest to declare.

BSLBs are versatile tools for studying the particulate output of T cells stimulated with model APC membranes. The flexibility of the method allows the reconstitution of complex and reductionist membrane compositions to study the effects of ligands and their signals on the secretion of tSVs and supramolecular attack particles and their components. We have tested this technology on various T cells, including preactivated TH, CTL, Tregs, and CART15. This protocol also works for the measurement of syn...

The immunological synapse (IS) is a pivotal molecular structure formed at the interface of cells engaged in physical contact that facilitates the regulated exchange of juxtracrine information. Different ISs have been described in the literature, and a growing body of evidence suggests these molecular hubs are a conserved feature of cellular networks. Various immune cells, including B cells, natural killer cells, dendritic cells, macrophages, and T cells, exchange information via the assembly of short-lived contacts1. Multiomic studies are advancing the understanding of novel subsets of leukocytes and stromal cells driving pathogenic cellular networks and expressing surface proteins with unknown functions. As synthetic APCs, BSLBs allow the direct investigation of the functional role of individual proteins in the integration of activating signals, namely antigens and costimulation/corepression, by T cells and the resulting release of effector particles referred to as signal four.
This paper describes the protocols and critical technical points to consider while using BSLBs to mimic the surface composition of model APCs. The protocols for the quantitative measurement of immune receptors and other surface proteins on APCs are presented along with the protocol for the reconstitution of synthetic APCs containing these measured quantities. Then, the steps required for coculturing T cells and BSLB are presented along with the protocol for the quantitative measurement of trans-synaptic particle transfer using flow cytometry. Most remarkably, BSLBs facilitate studying a plasma membrane-derived population of tSVs termed synaptic ectosomes (SEs). T-cell antigen receptor-enriched (TCR+) SEs are shed in response to TCR triggering2 and efficiently captured by BSLBs3, representing an excellent readout to assess the agonistic properties of antigens and the modelled membrane composition. CD63+ exosomes and supramolecular attack particles (SMAPs) are also released by stimulated T cells and captured by BSLBs. They can be used as additional readouts of activation and the resulting exocytic and lytic granule secretion by T cells. The mobilization of exocytic vesicles to the interacting pole of the T cell also facilitates the directional release of cytokines, such as IL-2, IFN-&#947;, and IL-10 in response to activation4,5,6,7,8. Although T-cell released cytokines can also be detected on BSLBs, a more dedicated study is currently under development to validate the quantitative analysis of cytokine release at the immunological synapse.
To interrogate how specific membrane compositions influence T cells&#39; synaptic output requires defining the physiological density of the target membrane component. Flow cytometry-based quantifications of cell surface proteins are an essential step in this protocol and require: 1) the use of antibodies with known numbers of fluorochromes per antibody (F/P), and 2) benchmark beads providing a standard reference for interpolating fluorochrome molecules from measured mean fluorescence intensities (MFIs).
These benchmark standards consist of five bead populations, each containing an increasing number of equivalent soluble fluorochromes (MESFs), which span the dynamic range of arbitrary fluorescence detection. These standard populations yield discrete fluorescence peaks, facilitating the conversion of arbitrary fluorescence units into MESFs by simple linear regression. The resulting MESFs are then used alongside antibody F/P values to calculate the average number of bound molecules per cell (or BSLB in later steps). The application of estimated cell surface areas to the average number of detected molecules then enables the calculation of physiological densities as molecules/&#181;m2. This quantification protocol can also be adapted to the measurement of protein densities on T cells and the biochemical reconstitution of membrane compositions mediating the formation of homotypic T cell synapses (i.e., T-T synapses9). If needed, the valency of antibody binding can be further estimated by using recombinant targets labeled with known numbers of fluorochromes per molecule. Then, the antibody-binding valency can be calculated for the same BSLB population by simultaneously comparing the number of bound fluorescent proteins and quantification antibodies (using two different quantification fluorochromes and MESF standards).
The reconstitution of APC membranes requires the assembly of supported lipid bilayers (SLBs) on silica beads1. Liposome stocks containing different phospholipid species can be harnessed to form a versatile lipid-bilayer matrix, enabling the anchoring of recombinant proteins with different binding chemistry (the preparation of liposomes is detailed in 10). Once the physiological density (or densities) of the relevant ligand &#34;on cells&#34; is defined, the same flow cytometry protocol is adapted to estimate the concentration of recombinant protein needed to coat BSLBs with the target physiological density. Two different anchoring systems can be used either in combination or separately.
First, SLB containing a final 12.5 mol% of Ni2+-containing phospholipids is sufficient to provide approximately 10,000 His-tag binding sites per square micron10 and works well to decorate BSLBs with most commercially available proteins whose physiological densities do not exceed this maximum loading capacity. The second loading system harnesses biotin-containing phospholipids (as mol%) to load biotinylated anti-CD3e Fab (or HLA/MHC monomers) via streptavidin bridges. The combination of these two BSLB decoration methods then enables the flexible tailoring of BSLBs as synthetic APCs. For highly complex APC surface compositions, the mol% of phospholipids and proteins can be increased to load as many proteins as the question at hand requires. Once the working concentrations of proteins and mol% of biotinylated phospholipids are defined, BSLBs can be assembled to interrogate the synaptic output of T cells with multiparametric flow cytometry.

<table><tbody><tr><td>1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)</td><td>Avanti Polar Lipids</td><td>790404C-25mg</td><td>18:1 DGS-NTA(Ni) in chloroform</td></tr><tr><td>&lt;br/&gt; PIPETMAN L Multichannel P8x200L, 20-200 &amp;micro;L</td><td>Gilson</td><td>FA10011</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phosphocholine</td><td>Avanti Polar Lipids</td><td>850375C-25mg</td><td>&amp;nbsp;18:1 (&amp;Delta;9-Cis) PC (DOPC) in chloroform</td></tr><tr><td>1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 390</td><td>ATTO-TEC</td><td>AD 390-165</td><td>DOPE ATTO 390</td></tr><tr><td>1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 488</td><td>ATTO-TEC</td><td>AD 488-165</td><td>DOPE ATTO 488</td></tr><tr><td>1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) ATTO 565</td><td>ATTO-TEC</td><td>AD 565-165</td><td>DOPE ATTO 565</td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt)</td><td>Avanti Polar Lipids</td><td>870273C-25mg</td><td>18:1 Biotinyl Cap PE in chloroform</td></tr><tr><td>200 &amp;micro;L yellow tips 10 x 96 Tips, Stack</td><td>Starlab</td><td>S1111-0206</td><td></td></tr><tr><td>5 mL polystyrene round-bottom tubes</td><td>Falcon&amp;reg;</td><td>352052</td><td></td></tr><tr><td>5.00 &amp;plusmn; 0.05 &amp;micro;m non-functionalized silica beads</td><td>Bangs Laboratories Inc.</td><td>SS05003</td><td></td></tr><tr><td>96 Well Cell Cultture Plate U-bottom with Lid, Tissue culture treated, non-pyrogenic.</td><td>Costar&amp;reg;</td><td>3799</td><td>For FCM staining and co-culture of BSLB and cells.</td></tr><tr><td>96 Well Cell Cultture Plate V-bottom with Lid, Tissue culture treated, non-pyrogenic.</td><td>Costar&amp;reg;</td><td>3894</td><td>For FCM staining of cells or beads in suspension.</td></tr><tr><td>Alexa Fluor 488 NHS Ester (Succinimidyl Ester)</td><td>Thermo Fisher Scientific, Invitrogen&amp;trade;</td><td>A20000</td><td></td></tr><tr><td>Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td><td>Thermo Fisher Scientific, Invitrogen&amp;trade;</td><td>A37573 and A20006</td><td></td></tr><tr><td>Allegra X-12R Centrifuge</td><td>Beckman Coulter</td><td></td><td>For normal in tube staining of biological samples for FCM</td></tr><tr><td>Aluminum Foil</td><td>Any brand</td><td></td><td>For protecting cells and BSLBs from light</td></tr><tr><td>anti-human CD154 (CD40L), clone 24-31</td><td>BioLegend</td><td>310815 and 310818</td><td>Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.</td></tr><tr><td>anti-human CD185 (CXCR5) Brilliant Violet 711, clone J252D4</td><td>BioLegend</td><td>356934</td><td>For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E</td></tr><tr><td>anti-human CD19 Brilliant Violet 421, clone HIB19</td><td>BioLegend</td><td>302234</td><td>For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E</td></tr><tr><td>anti-human CD2, clone RPA-2.10</td><td>BioLegend</td><td>300202</td><td>Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD2, clone TS1/8</td><td>BioLegend</td><td>309218</td><td>Brilliant Violet 421 conjugate.</td></tr><tr><td>anti-human CD252 (OX40L), clone 11C3.1</td><td>BioLegend</td><td></td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD28, clone CD28.2</td><td>eBioscience</td><td>16-0289-85</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD317 (BST2, PDCA-1), clone 26F8</td><td>ThermoFisher Scientific, invitrogen</td><td>53-3179-42</td><td>Alexa Fluor 488 conjugate, we found this clone to be cleaner than clone RS38E.</td></tr><tr><td>anti-human CD38, clone HB-7</td><td>BioLegend</td><td>356624</td><td>Alexa Fluor 700 conjugate</td></tr><tr><td>anti-human CD38, clone HIT2</td><td>BioLegend</td><td>303514</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD39, clone A1</td><td>BioLegend</td><td></td><td>Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD4 Brilliant Violet 650, clone OKT4</td><td>BioLegend</td><td>317436</td><td>For quantitative FCM analysis of tonsillar cells as shown in Fig. 1E</td></tr><tr><td>anti-human CD4, clone A161A1</td><td>BioLegend</td><td>357414 and 357421</td><td>PerCP/Cyanine5.5 and Alexa Fluor 647 conjugates, respectively</td></tr><tr><td>anti-human CD4, clone OKT4</td><td>BioLegend</td><td>317414 and 317422</td><td>PE/Cy7 and Alexa Fluor 647 conjugates, respectively</td></tr><tr><td>anti-human CD40, clone 5C3</td><td>BioLegend</td><td>334304</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD40, clone G28.5</td><td>BioLegend</td><td></td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD45, clone HI30</td><td>BioLegend</td><td>304056 and 368516</td><td>Alexa Fluor 647 and APC/Cy7 conjugates</td></tr><tr><td>anti-human CD47, clone CC2C6</td><td>BioLegend</td><td>323118</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD54 (ICAM-1), clone HCD54</td><td>BioLegend</td><td>322702</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD63 (LAMP-3), clone H5C6</td><td>BioLegend</td><td>353020 and 353015</td><td>PerCP/Cyanine5.5 and Alexa Fluor 647 conjugates, respectively</td></tr><tr><td>anti-human CD73, clone AD2</td><td>BioLegend</td><td>344002</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human CD80, clone 2D10</td><td>BioLegend</td><td>305216</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD81, clone 5A6</td><td>BioLegend</td><td>349512 and 349502</td><td>PE/Cy7 conjugate and labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester), respectively.</td></tr><tr><td>anti-human CD82, clone ASL-24</td><td>BioLegend</td><td>342108</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD86, clone IT2.2</td><td>BioLegend</td><td>305416</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human CD8a, clone HIT8a</td><td>BioLegend</td><td>300920</td><td>Alexa Fluor 700 conjugate</td></tr><tr><td>anti-human CD8a, clone SK1</td><td>BioLegend</td><td>344724</td><td>Alexa Fluor 700 conjugate</td></tr><tr><td>anti-human HLA-A/B/C/E, clone w6/32</td><td>BioLegend</td><td>311414 and 311402</td><td>Alexa Fluor 647 conjugate and Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human HLA-DR, clone L243</td><td>BioLegend</td><td>307656 and 307622</td><td>Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.</td></tr><tr><td>anti-human ICAM-1, clone HCD54</td><td>BioLegend</td><td>322702</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human ICOS, clone C398.4A</td><td>BioLegend</td><td>313516</td><td>Armenian Hamster IgG</td></tr><tr><td>anti-human ICOSL, clone MIH12</td><td>BioLegend</td><td>329611</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human ICOSL, clone MIH12</td><td>eBioscience</td><td>16-5889-82</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human LFA-1, clone TS1/22</td><td>BioLegend</td><td>Produced in house</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human OX40, clone Ber-ACT35 (ACT35)</td><td>BioLegend</td><td>350018</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human PD-1 , clone EH12.2H7</td><td>BioLegend</td><td>135230 and 329902</td><td>Alexa Fluor 647 conjugate and Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human PD-L1, clone 29E.2A3</td><td>BioLegend</td><td>329702</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human PD-L2, clone 24F.10C12</td><td>BioLegend</td><td>329611</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>anti-human PD-L2, clone MIH18</td><td>BioLegend</td><td>345502</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>anti-human TCRab, clone IP26</td><td>BioLegend</td><td>306712 and 306714</td><td>Alexa Fluor 488 and Alexa Fluor 647 conjugates, respectively.</td></tr><tr><td>antti-human CD156c (ADAM10), clone SHM14</td><td>BioLegend</td><td>352702</td><td></td></tr><tr><td>antti-human CD317 (BST2, Tetherin), clone RS38E</td><td>BioLegend</td><td>348404</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>Armenian Hamster IgG Alexa Fluor 647 Isotype control, clone HTK888</td><td>BioLegend</td><td>400902</td><td>Labelled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>BD Cytometer Setup and Tracking beads</td><td>Becton Dickinson &amp;amp; Company (BD)</td><td>641319</td><td>Performance track of instruments before quantitative FCM</td></tr><tr><td>BD FACSDiva</td><td>Becton Dickinson &amp;amp; Company (BD)</td><td>23-14523-00</td><td>Acquisition software</td></tr><tr><td>Bovine Seum Albumin</td><td>Merck, Sigma-Aldrich</td><td>A3294</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;, Calcium chloride</td><td>Merck, Sigma-Aldrich</td><td>C5670</td><td>anhydrous, BioReagent, suitable for insect cell culture, suitable for plant cell culture, &amp;ge;96.0%</td></tr><tr><td>Casein from bovine milk, suitable for substrate for protein kinase (after dephosphorylation), purified powder</td><td>Merck, Sigma-Aldrich</td><td>C5890</td><td></td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28</td><td>ThermoFisher Scientific, Gibco</td><td>11132D</td><td></td></tr><tr><td>DynaMag-2</td><td>ThermoFisher Scientific, Invitrogen&amp;trade;</td><td>12321D</td><td>For the removal of Dynabeads Human T-Activator CD3/CD28 in volumes less than 2 mL</td></tr><tr><td>DynaMag&amp;trade;-15</td><td>ThermoFisher Scientific, Invitrogen&amp;trade;</td><td>12301D</td><td>For the removal of Dynabeads&amp;trade; Human T-Activator CD3/CD28 in volumes less than 15 mL</td></tr><tr><td>Fetal Bovine Serum Qualified, One Shot</td><td>ThermoFisher Scientific, Gibco</td><td>A3160801</td><td>Needs heat inactivation for 30 min at 56 &lt;sup&gt;o&lt;/sup&gt;C</td></tr><tr><td>Ficoll-Paque PLUS</td><td>Cytiva, GE Healthcare</td><td>GE17-1440-02</td><td>Sterile solution of polysaccharide and sodium diatrizoate for lymphocyte isolation</td></tr><tr><td>Fixable Viability Dye eFluor 780</td><td>eBiosciences</td><td>65-0865-14</td><td>For the exclusion of dead cells during analyses</td></tr><tr><td>FlowJo</td><td>Becton Dickinson &amp;amp; Company (BD)</td><td>Version 10.7.1</td><td>Analysis software</td></tr><tr><td>Grant Bio MPS-1 Multi Plate Shaker</td><td>Keison Products</td><td>MPS-1</td><td>For the mixing of either cells during stainings or BSLBs during staning or protein loading (as an alternative to orbital agitation)</td></tr><tr><td>HEPES Buffer Solution (1 M)</td><td>ThermoFisher Scientific, Gibco</td><td>15630-056</td><td></td></tr><tr><td>HEPES, N-(2-Hydroxyethyl)piperazine-N&amp;prime;-(2-ethanesulfonic acid), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid.</td><td>Merck, Sigma-Aldrich</td><td>H4034</td><td>For preparation of HBS/HAS or HBS/BSA buffer. BioPerformance Certified, &amp;ge;99.5% (titration), suitable for cell culture</td></tr><tr><td>HERACell 150i CO&lt;sub&gt;2&lt;/sub&gt; incubator, 150 L, Electropolished Stainless Steel</td><td>ThermoFisher Scientific</td><td>51026282</td><td>For culturing and expanding purified CD4+ and CD8+ T cells.</td></tr><tr><td>Hula Mixer&amp;reg; Sample Mixer</td><td>ThermoFisher Scientific, Life Technologies</td><td>15920D</td><td>Vertical, variable-angle laboratory mixer used for the mixing of BSLBs and lipid master mix, blocking solutions, protein master mix and small scale antibody stainings.</td></tr><tr><td>Human Serum Albumin, 30% aqueous solution</td><td>Merck, Sigma-Aldrich</td><td>12667-M</td><td></td></tr><tr><td>Human TruStain FcX Fc Receptor Blocking Solution</td><td>BioLegend</td><td>422302</td><td>Fc Receptor Blocking Solution for blocking of Fc Receptors from biologically relevant samples</td></tr><tr><td>Innovatis CASY cell counter and analyzer TT</td><td>Biovendis Products GmbH</td><td></td><td>For the counting of cells and the determination of cell size and volume based on the exclusion of electric current.</td></tr><tr><td>KCl, Potassium chloride</td><td>Merck, Sigma-Aldrich</td><td>P5405</td><td>Powder, BioReagent, suitable for cell culture</td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>ThermoFisher Scientific, Gibco</td><td>25030-024</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;, Magessium chloride</td><td>Merck, Sigma-Aldrich</td><td>M2393</td><td>BioReagent, suitable for cell culture, suitable for insect cell culture</td></tr><tr><td>Microtube Insert for 24 x 1.5/2.0 mL tubes</td><td>Keison Products</td><td>P-2-24</td><td>Microtube insert for Grant Bio MPS-1 Multi Plate Shaker</td></tr><tr><td>Mini Incubator</td><td>Labnet International&amp;nbsp;</td><td>I5110A-230V</td><td>For the incubation (co-culturing) of BSLB and cells in the absence of CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Minimum Essential Medium Non-Essential Amino Acids</td><td>ThermoFisher Scientific, Gibco</td><td>11140-035</td><td></td></tr><tr><td>Mouse IgG polyclonal antibody control</td><td>Merck, Sigma-Aldrich</td><td>PP54</td><td>Used as positive control for the measurement of antibodies bound to mouse IgG capture bead standards</td></tr><tr><td>Mouse IgG1, k Isotype, clone MOPC-21</td><td>BioLegend</td><td>400129, 400112, 400130, 400144, 400128 and 400170</td><td>Alexa Fluor 488, PE, Alexa Fluor 647, Alexa Fluor 700, APC/Cyanine7 and Brilliant Violet 785&amp;nbsp; conjugates, respectively.</td></tr><tr><td>Mouse IgG1, k Isotype, clone X40</td><td>Becton Dickinson &amp;amp; Company (BD), Horizon</td><td>562438</td><td>Brilliant Violet 421 conjugate.</td></tr><tr><td>Mouse IgG1, &amp;kappa; Isotype control, clone P3.6.2.8.1</td><td>eBioscience</td><td>14-4714-82</td><td>Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>Mouse IgG2a, k Isotype, clone MOPC-173</td><td>BioLegend</td><td>400240</td><td>Alexa Fluor 647 conjugate</td></tr><tr><td>Mouse IgG2b, k Isotype, clone MPC-11</td><td>BioLegend</td><td>400330 and 400355</td><td>Alexa Fluor 647 and Brilliant Violet 785 conjugates, respectively</td></tr><tr><td>Multiwell 6 well Tissue culture treated with vacuum gas plasma</td><td>Falcon</td><td>353046</td><td>For culturing and expanding purified CD4&lt;sup&gt;+&lt;/sup&gt; and CD8&lt;sup&gt;+&lt;/sup&gt; T cells.</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, Disodium Phosphate</td><td>Merck, Sigma-Aldrich</td><td>S7907</td><td></td></tr><tr><td>NaCl, Sodium chloride</td><td>Merck, Sigma-Aldrich</td><td>S5886</td><td>BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, &amp;ge;99%</td></tr><tr><td>NiSO&lt;sub&gt;4&lt;/sub&gt;, Nickel(II) sulfate</td><td>Merck, Sigma-Aldrich</td><td>656895</td><td>For saturating NTA sites; added during the blocking process</td></tr><tr><td>Penicillin Streptomycin [+]10,000 units Penicillin; [+] 10,000 &amp;micro;g/mL Streptomycin</td><td>ThermoFisher Scientific, Gibco</td><td>15140-122</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4, sterile</td><td>ThermoFisher Scientific, Gibco</td><td>10010</td><td>No Ca&lt;sup&gt;2+&lt;/sup&gt; or Mg&lt;sup&gt;2+&lt;/sup&gt; added</td></tr><tr><td>Polyethersulfone (PES) Filter unit</td><td>Thermo Scientific Nalgene</td><td>&amp;nbsp;UY-06730-43</td><td>Hydrophilic PES membrane with low protein binding facilitates the filtering of solutions with high protein content</td></tr><tr><td>PURESHIELD argon ISO 14175-I1-Ar</td><td>BOC Ltd.</td><td>11-Y</td><td>For the protection of lipid stocks stored at +4 &amp;ordm;C.</td></tr><tr><td>Purified Streptavidin</td><td>BioLegend</td><td>280302</td><td></td></tr><tr><td>Quantum Alexa Fluor 488 MESF beads</td><td>Bangs Laboratories Inc.</td><td>488</td><td>Benchmark beads for the interpolation of Alexa Fluor 488 molecules bound to cells and/or BSLB</td></tr><tr><td>Quantum Alexa Fluor 647 MESF beads</td><td>Bangs Laboratories Inc.</td><td>647</td><td>Benchmark beads for the interpolation of Alexa Fluor 647 molecules bound to cells and/or BSLB</td></tr><tr><td>Rat anti-mouse IgG Kappa Light Chain, clone OX-20</td><td>ThermoFisher Scientific, invitrogen</td><td>SA1-25258</td><td>Labeled in house with Alexa Fluor 647 NHS Ester (Succinimidyl Ester)</td></tr><tr><td>Recombinant human IL-2</td><td>Peprotech</td><td>200-02-1MG</td><td></td></tr><tr><td>RMPI Medium 1640 (1x); [-] L-Glutamine</td><td>ThermoFisher Scientific, Gibco</td><td>31870-025</td><td></td></tr><tr><td>Rosette Human B Cell Enrichment Cocktail</td><td>STEMCELL Technologies</td><td>15064</td><td>Isolation of B cells for measuring densities of proteins in purified cell populations</td></tr><tr><td>Rosette Human CD4+ T Cell Enrichment Cocktail</td><td>STEMCELL Technologies</td><td>15022C.1</td><td></td></tr><tr><td>Rosette Human CD4+CD127low T Cell Enrichment Cocktail</td><td>STEMCELL Technologies</td><td>15361</td><td>Pre-enrichment of CD4+ CD127&lt;sup&gt;Low&lt;/sup&gt; T cells for the downstream isolation of Tregs by FACS.</td></tr><tr><td>Rosette Human CD8+ T Cell Enrichment Cocktail</td><td>STEMCELL Technologies</td><td>15063</td><td></td></tr><tr><td>RPMI Medium 1640 (1x); [-] Phenol Red</td><td>ThermoFisher Scientific, Gibco</td><td>11835-063</td><td>For the incubation (co-culturing) of BSLB and cells in the absence of CO&lt;sub&gt;2&lt;/sub&gt;. Phenol red-free media reduces the autofluorescence of cells in flow cytometry and microscopy based measurements.</td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>ThermoFisher Scientific, Gibco</td><td>11360-070</td><td></td></tr><tr><td>Sprout mini centrifuge</td><td>FisherScientific, Heathrow Scientific LLC</td><td>120301</td><td>Benchtop microcentrifuge used to wash silica beads and BSLB in 1.5 mL Eppendorf tubes.</td></tr><tr><td>Sterile cappeed 5 mL polystyrene round-bottom tubes</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>UltraComp eBeads Compensation Beads</td><td>ThermoFisher Scientific, invitrogen</td><td>01-2222-42</td><td></td></tr><tr><td>Zeba Spin Desalting Columns 7K MWCO</td><td>Thermo Fisher Scientific, Invitrogen&amp;trade;</td><td>89882</td><td></td></tr></tbody></table>

preparation of bead-supported lipid bilayers to study the particulate output of t cell immune synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

FCM for absolute protein quantification on the cell surface 
The reconstitution of BSLBs presenting physiological densities of ligands requires the estimation of total protein densities on the modeled cell subset. To reconstitute BSLBs, include any relevant ligand expected to play a role in the signaling axis of interest alongside proteins supporting the adhesion and functional interaction between BSLB and cells, such as ICAM-1 and costimulatory molecules, e.g., CD40, CD58, and B7 receptors (CD80 an...

Watch this Scientific Journal Video about Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses at JoVE.com

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

preparation-bead-supported-lipid-bilayers-to-study-particulate-output

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

4.1K Views.  The University of Oxford. Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells. 

Video: Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Biology

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Bioengineering

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Neuroscience

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres

Source: Curtis, B. N. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022).
This video describes the technique of studying the septin protein assembly on microspheres coated with lipid bilayers. This technique helps in understanding the curvature-dependent assemblies of protein on curved surfaces.

Source: Curtis, B. N. et al., Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022).
This ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

Utilizing Bead-Supported Lipid Bilayers to Investigate the Synaptic Output from T Cells

Source: C&#233;spedes, P. F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/63130">Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses.</a> J. Vis. Exp. (2022)
This video illustrates the reconstitution of synthetic antigen-presenting cells through Bead-Supported Lipid Bilayers and their application in assessing the synaptic output generated by activated T cells.

1. Protein density calibrations on BSLBs (Bead-supported lipid bilayers)

	Before taking the 5.00 &#177;0.05 &#181;m diameter non-functionalized silica ...

Immunology

Immunodiagnostics

Specialized Assays

An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers

Begin by using polypropylene scissor-type forceps to submerge glass coverslips in freshly prepared acidic piranha solution for 25 to 30 minutes. At the end of the wash, rinse the coverslips seven times in a fresh volume of ultra-pure water for each wash, and set the wet coverslips aside to let the remaining water roll off the clean glass.
When the coverslips are dry, aliquot two microliters of the final liposome mixture, precisely, in the center of a self-adhesive slide channel within a sterile flow hood, and immediately but carefully, align one clean and dry coverslip with the slide to allow the coverslip to be gently lowered onto the sticky side of the slide, and use the outer ring of the polypropylene scissor-type forceps to apply gentle pressure to the peripheral contact of the coverslip with the slide, making sure that the slip is tightly attached to the slide to prevent leakage. Then, turn the slide over, and use a permanent marker to draw four dots around the bilayer on the external side of the slide assembly.
Before the first injection, designate one port of the channel as the entry port and the other as the exit port, maintaining this designation throughout the experiment. To avoid bubble formation, insert the end of the pipette tip directly into the entry port until you feel the rubber seal of the slide channel being penetrated with the tip.
Slowly, fill the channel with 50 microliters of warm assay buffer. Inject 200 microliters of nickel(II) chloride in casein-blocking solution into the entry port, and remove 50 microliters of the buffer from the exit port. After a 45-minute incubation, remove the excess casein solution from the exit port.
Inject 100 microliters of an ICAM-1 and streptavidin solution into the entry port for a 45-minute incubation at room temperature. At the end of the incubation, remove the excess protein solution from the exit port, and wash the bilayer two times with 100 microliters of assay buffer per wash. Then, label the bilayers with 100 microliters of fluorophore-conjugated anti-CD3 antibody for 45 minutes at room temperature, followed by two washes in 100 microliters of assay buffer per wash, as demonstrated.
To image T cell-bilayer interactions, place the slide on the 37-degree Celsius heated stage of a Total Internal Reflection Fluorescence, or TIRF, microscope, and adjust the stage using the ink marks to center the bilayer onto the 100 power objective.
For granule release imaging, add fluorescence-conjugated anti-CD107a antibody Fab fragments to the CD4 T cell suspension, and resuspend the labeled cells in 50 microliters of assay buffer for injection into the entry port of the slide channel. Then, select the appropriate number of experimental fields, and record images of each field once every minute for 30 minutes after the injection.