Name: Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses (Video) | JoVE Journal
Uploaded: 2022-04-01T13:30:00.000+00:00
Duration: 11 min 6 s
Description: Learn to prepare bead-supported lipid bilayers for studying T cell immune synapse output using flow cytometry. This protocol enables quantitative analysis of trans-synaptic particles in immunological research.

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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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<li>Nguyen, R., Perfetto, S., Mahnke, Y. D., Chattopadhyay, P., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+spillover+spreading+for+comparing+instrument+performance+and+aiding+in+multicolor+panel+design%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design.</a> Cytometry A. 83 (3), 306-315 (2013).</li>
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<li>Yoon, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+non-activated+lymphocytes+using+three-dimensional+refractive+index+tomography+and+machine+learning%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Identification of non-activated lymphocytes using three-dimensional refractive index tomography and machine learning.</a> Scientific Reports. 7 (1), 6654(2017).</li>
<li>Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distributions+of+autofluorescence+after+compensation%3A+Be+panglossian%2C+fret+not%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Distributions of autofluorescence after compensation: Be panglossian, fret not.</a> Cytometry A. 89 (4), 398-402 (2016).</li>
<li>Maecker, H. T., Trotter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+controls%2C+instrument+setup%2C+and+the+determination+of+positivity%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Flow cytometry controls, instrument setup, and the determination of positivity.</a> Cytometry A. 69 (9), 1037-1042 (2006).</li>
<li>Balint, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Supramolecular+attack+particles+are+autonomous+killing+entities+released+from+cytotoxic+T+cells%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Supramolecular attack particles are autonomous killing entities released from cytotoxic T cells.</a> Science. 368 (6493), 897-901 (2020).</li>
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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

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 and ...

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

Research

JoVE Journal

Immunology and Infection

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. <br...

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

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.

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

This video demonstrates a technique for visualizing the formation of the immunological synapse in natural killer (NK) cells on a supported lipid bilayer (SLB). The SLB is first labeled with a fluorophore-tagged NK cell activating receptor ligand, followed by incubation with NK cells. Subsequently, the cells are stained for polymerized actin and perforin-positive lytic granules. This approach enables the visualization of synapse formation using stimulated emission depletion (STED) microscopy.

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

This video demonstrates an in vitro technique to study immune synapse formation between primary human T cells and supported lipid bilayers (SLB). T cell surface proteins interact with respective ligands immobilized on SLB to form an immune synapse, followed by the movement of lytic granules towards the synapse, which is visualized using fluorescence microscopy.

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

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.

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

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.

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

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.

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

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

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.

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

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.

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

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.

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

Learn to prepare bead-supported lipid bilayers for studying T cell immune synapse output using flow cytometry. This protocol enables quantitative analysis of trans-synaptic particles in immunological research.

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

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