Analysis of Immune Synapses between Human T Cells and SEB-Loaded Raji Cells using Imaging Flow Cytometry

Abstract

Source:  Wabnitz, G. et al., Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry. J. Vis. Exp. (2019)

This video analyzes the immune synapse formation between T cells in unpurified pan-leukocyte preparations and Staphylococcus aureus enterotoxin B, SEB-loaded Raji cells. CD3 and F-actin enrichment is observed in the immune synapse using imaging flow cytometry.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Preparation of Pan-leukocytes

1. Draw 1 mL of peripheral blood from a healthy donor (or patient) in a heparinized syringe. Make sure to have approval by the responsible ethics committee for the blood donation.
2. Mix 1 mL of human peripheral blood with 30 mL of ACK lysis buffer (150 mM NH₄Cl, 1 mM KHCO₃, and 0.1 mM EDTA, pH 7.0) in a 50 mL tube and incubate for 8 min at room temperature.
3. Fill the tubes with PBS and centrifuge at 300 x g....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Multifuge 3 SR	Heraeus
RPMI 1640	LifeTechnologies	#11875085	500 mL
FCS	Pan Biotech #3302-P101102
Polystyrene Round-Bottom Tube	Falcon	#352054	5 mL
Dulbecco's Phosphate-Buffered Saline	Sigma	D8662
Bovine Serum Albumin	Roth	#8076.3
Saponin	Sigma	S7900
Paraformaldehyde	Sigma Aldrich	#16005
Speed Bead	Amnis	#400041
Minishaker MS1	IKA Works	MS1
Microtiter plate	Greiner Bio One	#650101	96U
Enterotoxin SEB	Sigma Aldrich	S4881
DAPI	Sigma Aldrich	D9542	One to three thousand ratio
CD3-PeTxR	Invitrogen	MHCD0317	One to thirty ratio
Phalloidin-AF647	Molecular Probes	A22287	One to one fifty ratio
IS100	Amnis		Imaging flow cytometer
IDEAS	Amnis		Software
INSPIRE	Amnis		Software