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…
Disclosures
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 B | 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 |
Tags
Cite This Article
Assessing Immunological Synapse Topology through Live-Cell Imaging. J. Vis. Exp. (Pending Publication), e21890, doi: (2024).