Assessing Immunological Synapse Topology through Live-Cell Imaging

Published: January 31, 2024

Abstract

Source: Martinelli, R. et al., An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics. J. Vis. Exp. (2015)

This video demonstrates live-cell imaging of immunological synapse topology, investigating the interactions between T lymphocytes and epithelial cells carrying fluorescent antigens. Fluorescence microscopy reveals red cytoplasm displacement and yellow membrane rings in endothelial cells, indicating the formation of immunological synapses.

Protocol

1. Preparing Human CD4+ Th1 Effector/Memory T Cells Apply a tourniquet to the arm of the donor, wipe the vein with alcohol, and insert a needle. Slowly draw 15 ml of blood into a vacutainer using EDTA as an anticoagulant. When the blood has been drawn, untie the tourniquet before removing the needle. Immediately apply pressure to the wound with sterile gauze when the needle is removed. Transfer blood to a 50 ml tube. Add RPMI-1640 at RT at a 1:1 dilution (final volum…

Divulgazioni

The authors have nothing to disclose.

Materials

BD Vacutainer stretch latex free tourniquet BD Biosciences 367203
BD alcohol swabs BD Biosciences 326895
BD Vacutainer Safety-Lok BD Biosciences 367861 K2 EDTA
BD Vacutainer Push Button Blood Collection Set BD Biosciences 367335
RPMI-1640 Sigma-Aldrich R8758-1L
Ficoll-Paque  Sigma-Aldrich GE17-1440-02 Bring to RT before use
FCS-Optima Atlanta Biologics s12450 Heat inactivated
Penicillin-Streptomycin  Sigma-Aldrich  P4458-100ML  
Trypan blue Sigma-Aldrich T8154-20ML
Staphylococcal enterotoxin Toxin Technology BT202RED Stock solution 1mg/ml in PBS
Toxic shock syndrome toxin 1  Toxin Technology TT606RED Stock solution 1mg/ml in PBS
Human IL-15 R&D Systems 247-IL-025 Stock solution 50ug/ml in PBS
PBS Life Technologies 10010-049
Fibronectin Life Technologies 33016-015 Stock solution 1mg/ml in H20
HMVEC-d Ad-Dermal MV Endo Cells Lonza CC-2543 Other Human Microvascular ECs can be used, i.e. HLMVECs
EGM-2 MV bullet kit Lonza CC-3202
Trypsin-EDTA Sigma-Aldrich T-4174 Stock solution 10x, dilute in PBS
Amaxa-HMVEC-L Nucleofector Kit Lonza vpb1003 Required Kit for step 4
IFN-g Sigma-Aldrich I3265 Stock solution 1mg/ml in H20
TNF-alpha 10ug, human Life Technologies PHC3015 Stock solution 1mg/ml in H20
Phenol Red-free HBSS  Life Technologies 14175-103
Hepes Fisher Scientific BP299-100
Calcium Chloride Sigma-Aldrich C1016-100G Stock solution 1M in H20
Magnesium chloride Sigma-Aldrich 208337 Stock solution 1M in H20
Human Serum albumin Sigma-Aldrich A6909-10ml
Immersol 518 F fluorescence free Immersion oil Fisher Scientific 12-624-66A
Fura-2 AM 20x50ug Life Technologies F1221 Stock solution 1mM in DMSO
pEYFP-Mem (Mem-YFP) Clontech 6917-1
pDsRed-Monomer (Soluble Cytoplasmic DsRed) Clontech 632466
pDsRed-Monomer Membrane (Mem-DsRed) Clontech 632512
pEGFP-Actin Clontech 6116-1
Alexa Fluor 488 Phalloidin Life Technologies A12379
 Falcon 15mL Conical Centrifuge Tubes Fisher Scientific 14-959-70C
Falcon 50mL Conical Centrifuge Tubes Fisher Scientific 14-959-49A 
Falcon Tissue Culture Treated Flasks T25 Fisher Scientific 10-126-9
 Falcon Tissue Culture Treated Flasks T75 Fisher Scientific   13-680-65
 Corning Cell Culture Treated T175 Fisher Scientific 10-126-61 
Glass coverslips  Fisher Scientific 12-545-85  12 mm diameter
 Falcon Tissue Culture Plates 24-well Fisher Scientific 08-772-1
Delta-T plates Bioptechs 04200415B
Wheaton Disposable Pasteur Pipets Fisher Scientific 13-678-8D
1.5 ml Eppendorf tube  Fisher Scientific 05-402-25
 ICAM1 mouse anti-human BD Biosciences 555509
HS1 mouse anti-human BD Biosciences 610541
Anti-Human CD11a (LFA-1alpha) Purified ebioscience BMS102
Anti-Human CD3 Alexa Fluor® 488 ebioscience 53-0037-41
Anti-MHC Class II antibody  Abcam ab55152
Anti-Talin 1 antibody Abcam ab71333
Anti-PKC theta antibody  Abcam ab109481
Phosphotyrosine (4G10 Platinum) Millipore 50-171-463
Nucleofector II Amaxa Biosystems Required electroporator for step 4
Zeiss Axiovert Carl Zeiss MicroImaging
Zeiss LSM510  Carl Zeiss MicroImaging
Zeiss Axiovison Software Carl Zeiss MicroImaging
NU-425 (Series 60) Biological Safety Cabinet NuAIRE Nu-425-600
 Forma STRCYCLE 37 °C, 5% CO2 Cell culture Incubator Fisher Scientific 202370
Centrifuge 5810 Eppendorf EW-02570-02
Hemocytometer Sigma-Aldrich  Z359629 Bright-Line Hemocytometer
Isotemp Waterbath model 202 Fisher Scientific 15-462-2

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Citazione di questo articolo
Assessing Immunological Synapse Topology through Live-Cell Imaging. J. Vis. Exp. (Pending Publication), e21890, doi: (2024).

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