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Flow Cytometry

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells.

Alex Shepherd^1,2, Bigitha Bennychen^1,2, Zafer Ahmed¹

¹Human Health Therapeutics Research Centre, National Research Council, Ottawa, ON, Canada.

|July 1, 2024

Summary

This summary is machine-generated.

This study introduces a simple flow cytometry assay to measure chimeric antigen receptor (CAR) T-cell binding kinetics. The assay quantizes CAR-target cell interactions, aiding the understanding of CAR functionality in cancer therapy.

Keywords:

CAR-T Cell-to-cell interaction Cellular avidity Cellular kinetics Flow cytometry High throughput Jurkat Screening T cell

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Published on: March 14, 2025

Area of Science:

Immunology
Biotechnology
Cell Biology

Background:

Chimeric antigen receptors (CARs) reprogram immune cells for cancer therapy, particularly in hematological malignancies.
Understanding the mechanisms and structural properties governing CAR responses is crucial for optimizing their efficacy.
Current knowledge gaps exist regarding the precise factors influencing CAR-mediated cellular interactions and therapeutic outcomes.

Purpose of the Study:

To develop and validate a straightforward assay for assessing cellular avidity of CAR-expressing cells.
To characterize the interaction kinetics between CAR-expressing T cells and target antigen-expressing cells.
To provide a tool for better understanding CAR functionality and signaling events.

Main Methods:

Utilized a standard flow cytometer to measure interaction kinetics between CAR-expressing Jurkat cells and target cells.
Employed co-culture of CAR Jurkat cells with target-positive and target-negative cells under varying effector-target ratios.
Observed the formation of CAR-target cell doublets as a readout of actively bound cells, without the need for staining.

Main Results:

Developed a simplified assay to quantify CAR-mediated cellular binding kinetics.
Demonstrated that binding curves varied between different CAR constructs based on antigen-binding domains.
Confirmed that cellular binding kinetics are consistent, antigen-specific, and require active biological signaling.

Conclusions:

The developed assay provides a simple, rapid method to assess CAR-mediated cellular avidity using flow cytometry.
This assay can be applied to various CAR targets, including those relevant to solid tumors and hematological cancers.
The protocol's ability to reveal unique binding curves aids in understanding CAR-target interactions and proximal signaling, potentially improving CAR-based therapies.