CAR expression assays verify that engineered T cells are expressing the CAR construct at high levels. Multiple methods are used to detect CAR expression, each addressing different aspects:

By combining these assays, researchers confirm that their CAR-T product is properly engineered (identity) and quantitatively assess how well the CAR is expressed on the cells. Each technique complements the others: for example, flow gives rapid single-cell data, qPCR offers detection beyond flow’s limits, Western validates protein size, and IHC reveals spatial distribution. AAT Bioquest supplies specialized reagents for all four methods, ensuring reliable and bright signals in CAR detection assays.

Cell viability assays determine what fraction of the CAR-T cells are alive and healthy (typically requiring ≥70% viability for a therapeutic dose). High viability is essential both to meet release criteria and to ensure downstream assays (proliferation, cytotoxicity) yield meaningful results. Fluorescence-based viability methods allow quick, multiparametric readouts of live vs. dead cells in CAR-T samples:

Together, these viability tools help confirm that CAR-T cells meet quality thresholds before use. They also facilitate troubleshooting of culture conditions or treatment effects by clearly distinguishing healthy cells from dying cells. AAT Bioquest’s viability reagents are designed for maximum signal clarity and ease-of-use, ensuring that live/dead status can be determined accurately alongside other phenotypic or functional markers.

Proliferation assays measure the ability of CAR-T cells to undergo repeated cell divisions and expand in number after activation. Robust proliferative capacity is a key indicator of a potent CAR-T product (it suggests the cells can expand in the patient to fight cancer). Two complementary fluorescence approaches are commonly used to evaluate CAR-T proliferation in vitro:

Using these methods, scientists can confirm that CAR-T cells not only survive but also vigorously expand upon antigen encounter. Proliferation readouts (like division index and %EdU⁺ cells) correlate with in vivo expansion potential. With AAT’s bright and resilient proliferation tracers and kits, even multiple rounds of cell division can be tracked clearly, supporting the development of CAR-T products that meet potency requirements for expansion.

Cytotoxicity assays evaluate how efficiently CAR-T cells recognize and kill antigen-expressing target cells. This is the core functional test of CAR-T potency. There are two major formats for assessing cytotoxicity:

By performing cytotoxicity assays across a range of E:T ratios or time points, researchers can benchmark the killing potency of their CAR-T cells. AAT’s flow-based and plate-based assay reagents ensure that labs have flexibility to measure cytotoxic function in whichever format suits their workflow, all while maintaining high sensitivity and avoiding hazardous radioisotopes. Reliable cytotoxicity data is often required for regulatory submissions, and these fluorescence assays provide that information with ease and precision.

Persistence assays evaluate the longevity and sustained function of CAR-T cells over an extended period. Long-term persistence is linked to durable patient remissions, so demonstrating this attribute is a critical part of CAR-T characterization. Key strategies include:

By combining in vitro and in vivo persistence assays, researchers gain a complete picture of CAR-T durability. If CAR-T cells continue dividing and killing over multiple challenges and can still be detected long after infusion, it suggests a therapy capable of lasting remission. AAT Bioquest’s long-term tracking reagents (proliferation dyes, NIR cell trackers, PCR detection kits, and IHC amplifiers) equip scientists to demonstrate this critical property with confidence.