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

Published: November 30, 2023

Abstract

Source: Steblyanko, M., et al. Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue. J. Vis. Exp. (2018).

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.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Isolation of CD4+ T Cells for an Image Analysis Thaw a 1 mL aliquot containing 107 frozen peripheral blood mononuclear cells (PBMCs) or lymph node mononuclear cells (LNMCs) from collected samples. In a sterile hood, add the thawed cells to 9 mL of RPMI supplemented with penicillin/streptomycin and glutamine. Cent…

Divulgations

The authors have nothing to disclose.

Materials

CD4 T cell isolation kit, human Miltenyl Biotec 130-096-533
CD8 T cells Isolation Kit, human Miltenyl Biotec 130-096-495
DOPC Avanti Polar Lipids 850375C
DOGS NTA Avanti Polar Lipids 790528C
Biotinyl Cap PE Avanti Polar Lipids 870273C
Human Serum Albumin Octapharma USA NDC 68982-643-01
sticky-Slide VI 0.4 ibidi 80608
Coverslips for sticky-Slides ibidi 10812
Bioptech FCS2 Chamber Bioptech 060319-2-03
anti-CD3 antibody Thermo Fisher Scientific 16-0037-81 OKT3 clone, hybridoma cells are available from ATCC
anti- CD28 antibody Genetex GTX14664 9.3 clone
Casein Sigma C5890
Biotin-PEO4-NHS Thermo Fisher Scientific 21329
DMSO Sigma D2650-5
Alexa Fluor 488 protein labeling kit with column for labeled protein purification Thermo Fisher Scientific A10235
Alexa Fluor 568 protein labeling kit with column for labeled protein purification Thermo Fisher Scientific A10238
Amersham Cy5 NHS Ester GE Life Science PA15101
pMT/V5-His A, B, C Drosophila Expression Vectors Thermo Fisher Scientific V412020
pcopneo, G418 Drosophila expression vector for positive selection ATCC 37409
Serum free Drosophial media Insect-XPRESS Lonza 12-730Q
Hybridoma YN1/1.7.4 ATCC CRL1878 The hybridoma secrets antibody against ICAM-1.
Cyanogen bromide-activated-Sepharose 4B Sigma-Aldrich C9142 Utilized for preparation of Sepharose with covelently bound anti-ICAM antibody.
MasterFlex tangential flow concentrator Cole-Parmer 77601-60 7592-40 Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant
Centramate Lab Tangential Flow Systems Pall Laboratory FS002K10 OS010T12 FS005K10 Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant
Ni-NTA Agarose QIAGEN 30210
Dialysis tubing Spectra/Por 131384
Papain from papaya latex Sigma P3125
mouse anti-human antibody against CD107a BD Bioscences 555798 Clone H4A3
Ansell Natural Blue Gloves Fisher Scientific 19-014-539
Nalgene Polypropylene Scissor-Type Forceps Thermo Fisher Scientific 6320-0010
Streptavidin ProZyme SA10
Confocal microscope Nikon Nikon TiE inverted microscope equipped with PFS for long-term image stability control, 60x oil objectives, 4 lasers with excitation lines at 405, 458, 488, 514, 561, and 640 nm, 2 GaAsP detectors and 2 high sensitivity PMTs, DIC transmitted light, Programmable X,Y,Z stage for multiple positions and stitching of large areas, time lapse functions, Tokai-Hit temperature and CO2-controlled chamber for live imaging, and anti-vibration isolation table
TIRF microscope Andor Andor Revolution XD system equipped with Nikon TIRF-E illuminator, Lasers with 405,488,561 and 640 lines, DIC transmitted light, Yokogawa CSU-X1 spinning disk head for confocal imaging, 100/1.49 NA objective, Andor iXon X3 EM-CCD camera, objective heater, and a piezoelectric motorized stage with Perfect Focus System (PFS)

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Citer Cet Article
An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers. J. Vis. Exp. (Pending Publication), e21852, doi: (2023).

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