High-dimensional functional phenotyping of preclinical human CAR T cells using mass cytometry

Ilaria M Michelozzi; Jahangir Sufi; Thomas A Adejumo; Persis J Amrolia; Christopher J Tape; Alice Giustacchini

doi:10.1016/j.xpro.2022.101174

High-dimensional functional phenotyping of preclinical human CAR T cells using mass cytometry

STAR Protoc. 2022 Feb 9;3(1):101174. doi: 10.1016/j.xpro.2022.101174. eCollection 2022 Mar 18.

Authors

Ilaria M Michelozzi¹, Jahangir Sufi², Thomas A Adejumo³, Persis J Amrolia^{1

4}, Christopher J Tape², Alice Giustacchini¹

Affiliations

¹ Molecular and Cellular Immunology Section, UCL Great Ormond Street Institute of Child Health, Zayed Centre For Research into Rare Disease in Children, WC1N 1DZ London, UK.
² Cell Communication Lab, Department of Oncology, University College London Cancer Institute, WC1E 6DD London, UK.
³ Fluidigm Corporation, South San Francisco, CA, USA.
⁴ Department of Bone Marrow Transplant, Great Ormond Street Hospital for Children, WC1N 3JH London, UK.

Abstract

Here, we present a comprehensive protocol for the generation and functional characterization of chimeric antigen receptor (CAR) T cells and their products by mass cytometry in a reproducible and scalable manner. We describe the production of CAR T cells from human peripheral blood mononuclear cells. We then detail a three-step staining protocol with metal-labeled antibodies and the subsequent mass cytometry analysis. This protocol allows simultaneous characterization of CAR T cell intracellular signaling, activation, proliferation, cytokine production, and phenotype in a single assay.

Keywords: Cell Biology; Cell culture; Cell isolation; Flow Cytometry/Mass Cytometry; Health Sciences; Immunology; Single Cell.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antibodies
Humans
Leukocytes, Mononuclear*
T-Lymphocytes*

Substances

Antibodies

Abstract

Publication types

MeSH terms

Substances

Grants and funding