Protocol for live-cell imaging of immune synapse formation and activation of CAR T cells against cancer cells

Jorge Ibanez; Nikhil Hebbar; Unmesha Thanekar; Zhongzhen Yi; Haley Houke; Meghan Ward; Chris Nevitt; Liqing Tian; Stephen C. Mack; Heather Sheppard; Jason Chiang; M. Paulina Velasquez; Giedre Krenciute

STAR Protocols (Dec 2024)

Protocol for live-cell imaging of immune synapse formation and activation of CAR T cells against cancer cells

Jorge Ibanez,
Nikhil Hebbar,
Unmesha Thanekar,
Zhongzhen Yi,
Haley Houke,
Meghan Ward,
Chris Nevitt,
Liqing Tian,
Stephen C. Mack,
Heather Sheppard,
Jason Chiang,
M. Paulina Velasquez,
Giedre Krenciute

Affiliations

Jorge Ibanez: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Nikhil Hebbar: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Unmesha Thanekar: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Zhongzhen Yi: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Haley Houke: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Meghan Ward: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Chris Nevitt: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Liqing Tian: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Stephen C. Mack: Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Heather Sheppard: Department of Pathology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
Jason Chiang: Department of Pathology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
M. Paulina Velasquez: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA; Corresponding author
Giedre Krenciute: Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA; Corresponding author

Journal volume & issue: Vol. 5, no. 4
p. 103422

Abstract

Read online

Summary: Immune synapse (IS) formation determines T cell antitumor activity. Here, we present a protocol for characterizing the IS formation between chimeric antigen receptor (CAR) T cells and tumor cells by measuring the IS size and calcium flux by live-cell imaging. We describe steps for CAR T cell manufacturing, sample preparation, image acquisition, and data analysis.For complete details on the use and execution of this protocol, please refer to Chockley et al.,1 Ibanez et al.,2 and Zoine et al.3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

About the journal

Abstract

Keywords