CAR-T Expression

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:

• Flow cytometry: Measures the percentage of T cells with CAR protein on their surface and the intensity of expression for each cell. It provides single-cell resolution and can be multiplexed with other markers (e.g. T cell phenotypes, viability). AAT’s iFluor® dye-conjugated antibodies and mFluor™ polymer tags deliver brighter, more photostable signals than standard dyes, enabling detection of even dim CAR levels.
• qPCR: Quantifies the CAR gene copies or mRNA in a sample, offering extremely high sensitivity. qPCR can detect rare CAR-T cells in a large background (useful for tracking persistence) and determines transgene copy number per cell. AAT provides optimized primer/probe sets and Helixyte™ nucleic acid quantitation kits to ensure precise CAR gene measurement.
• Western blot: Confirms the presence and approximate size of the CAR protein in cell lysates. This method is useful to verify protein integrity or detect intracellular epitopes. AAT’s ultra-sensitive Amplite™ chemiluminescent substrates and Buccutite™ poly-HRP reagents enhance detection of CAR bands, revealing low-abundance proteins that might be missed by traditional Western blot reagents.
• Immunohistochemistry (IHC): Detects CAR-T cells in tissue sections, showing their location in vivo. IHC (or immunofluorescence) uses anti-CAR antibodies on fixed tissues. AAT’s Power Styramide™ (PSA™) signal amplification kits and colorimetric substrates (e.g. StayRight™ Purple) markedly boost sensitivity, allowing visualization of rare CAR-T cells in tumors or organs with minimal background.

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.

Flow Cytometry & qPCR Reagents

Western Blot Reagents

Immunohistochemistry Reagents

Viability

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:

• Membrane integrity dyes: Use impermeant DNA-binding dyes to label dead cells with compromised membranes, while live cells exclude the dye. This is the standard live/dead discrimination in flow cytometry (e.g. PI, 7-AAD). AAT’s Cell Meter™ Fixable Viability Dyes improve on this approach by covalently tagging dead cells in a range of colors (UV through NIR-excited), making the staining robust through fixation and compatible with multicolor panels. These dyes yield brighter, more stable signals than traditional viability dyes, ensuring a clear separation of live and dead cell populations.
• Enzymatic activity markers: Detect esterase activity present only in live cells. For example, Calcein-AM enters all cells but is converted into fluorescent calcein (green) only by enzymes in viable cells. Dead cells, lacking esterase activity and with leaky membranes, remain non-fluorescent (or are counterstained by a second dye). AAT’s Live-or-Dead™ Viability/Cytotoxicity Kit uses Calcein-AM (live, green) in combination with a red nuclear dye for dead cells, providing a dual-color readout of live and dead cells in one step. This method is extremely sensitive and works for both flow cytometry and microscopy.
• No-wash, real-time viability dyes: These reagents continuously report cell viability without wash steps. AAT’s ReadiUse™ DXAq7™ is a ready-to-use analog of DRAQ7™ that can be added directly to CAR-T cultures or co-cultures. It emits a far-red nuclear fluorescence in cells as they lose membrane integrity, allowing kinetic monitoring of cell death. This dye is brighter than the original DRAQ7 and requires no fixation or removal, enabling real-time assessment of CAR-T viability (for example, during cytotoxicity assays) with minimal handling.

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.

Live/Dead Discrimination Reagents

Proliferation & Expansion

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:

• Dye dilution tracking: CAR-T cells are uniformly labeled with a cell-tracing dye before stimulation. As the cells divide, the dye is partitioned between daughter cells, causing fluorescence intensity to halve with each division. Flow cytometry then reveals distinct peaks corresponding to 0 divisions, 1 division, 2 divisions, etc., from which the proliferation history can be quantified. AAT’s CytoTell™ proliferation dyes (available in violet-excited, green, and red/NIR-excited versions) produce bright, even labeling that can track 6–8+ generations of divisions. These dyes are designed to minimize spectral overlap (e.g. avoiding the FITC/GFP channel), enabling use in CAR-T cells that express GFP or are combined with FITC-labeled antibodies. Their high stability and low toxicity ensure that labeling does not hinder the cells’ ability to proliferate.
• DNA synthesis (EdU incorporation): Actively dividing cells can also be identified by incorporating synthetic nucleosides during DNA replication. The EdU (5-ethynyl-2′-deoxyuridine) assay labels newly synthesized DNA, which is then detected by a fluorescent “click” reaction. AAT Bioquest’s BuccuLite™ XdU Flow Kits provide an EdU analog along with a copper-free click chemistry detection, allowing S-phase cells to be labeled without the cytotoxicity associated with copper reagents. By adding the EdU analog to a CAR-T culture for a short pulse (a few hours) and then staining, researchers can measure what fraction of the CAR-T population was in S-phase (actively replicating) during that period. Combining generation tracing and EdU incorporation offers a comprehensive view of CAR-T proliferative capacity (overall divisions and current cell cycle activity).

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.

Cell Proliferation Tracers & Kits

Cytotoxicity

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:

• Flow cytometry-based killing assays: In this approach, target cells are labeled with a distinct fluorescent dye and mixed with CAR-T effector cells at a defined effector:target (E:T) ratio. After co-incubation for a set period, the cells are stained with a viability dye (or apoptosis marker like Annexin V). Flow cytometry is then used to identify the target cell population (by the tracer dye) and determine what fraction of those targets are dead. This single-cell method provides a precise percentage of target cell lysis and can also reveal how each target died (for example, using Annexin V for apoptosis vs. a DNA dye for membrane lysis). AAT offers ideal tools for this method: stable CellTrace/CytoTell™ dyes to pre-label target cells, and bright viability dyes (7-AAD, propidium iodide, etc.) or Annexin V conjugates to stain dead targets. These reagents enable sensitive detection of specific killing even at low E:T ratios, without radioactive labels.
• Bulk (plate-based) cytotoxicity assays: These assays measure overall target cell viability or released contents in a mixed culture, typically in 96-well plates for higher throughput. After co-culturing CAR-T with target cells, the remaining viable target cells or the released biomarkers from lysed cells are quantified. One common approach is a resazurin assay: live cells convert resazurin to a fluorescent product (resorufin), so loss of fluorescence indicates target cell killing. Another is the LDH release assay: dead cells release lactate dehydrogenase (LDH) into the supernatant, which can be detected colorimetrically. AAT Bioquest’s Cell Meter™ fluorimetric cytotoxicity assay kit (resazurin-based) and Amplite™ LDH assay kit provide simple add-and-read solutions for these measurements. They are non-radioactive, sensitive, and compatible with standard plate readers. Using these kits, laboratories can generate cytotoxicity dose-response curves and calculate metrics like EC₅₀ (the effector cell concentration for 50% killing) efficiently, replacing the old ⁵¹Cr release assays.

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.

Target Cell Killing Assay Reagents

Persistence

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:

• Serial restimulation in vitro: CAR-T cells are repeatedly stimulated (for example, co-cultured with fresh target cells or stimulatory beads in cycles) over the course of days or weeks. Their ability to survive and continue proliferating with each stimulation is measured. Using AAT’s proliferation tracking dyes (CytoTell™ series), researchers can monitor cell division over multiple weeks, observing whether generation peaks continue to form as the cells keep dividing. Additionally, periodic viability or metabolic assays (such as resazurin-based readouts) can quantify how the CAR-T population grows or declines over time. A CAR-T product with strong persistence will maintain high viability and keep expanding (or at least remain stable) through successive stimulations, indicating resistance to exhaustion.
• In vivo tracking and imaging: To assess persistence in an animal model, CAR-T cells can be labeled and then followed after infusion. Near-infrared (NIR) membrane dyes like AAT’s DiR dye (a DiIC₁₈ carbocyanine) are ideal for this purpose: CAR-T cells are labeled with DiR and injected, and then non-invasive imaging (e.g. using an IVIS system) detects the NIR signal in the body over time. This reveals how long the cells survive and where they migrate (biodistribution). DiR’s deep-red fluorescence penetrates tissues well, enabling detection of CAR-T cells in organs. Using such imaging, studies have shown CAR-T cells homing to tumor sites or persisting in lymphoid organs for weeks. The strength and duration of the signal indicate the persistence of the cells in vivo.
• Highly sensitive ex vivo detection: Even if CAR-T cells drop to low levels in the body, advanced assays can find them. One approach is to take blood or tissue samples at intervals and test for CAR-T presence. Flow cytometry with ultra-bright CAR detection reagents (like iFluor® antibody conjugates or amplified staining) can sometimes identify rare CAR-T cells in a sample. More commonly, quantitative PCR is used to detect CAR-specific DNA sequences in blood or tissue, which can pick up even a few CAR-T cells. AAT supplies primer/probe sets for CAR transgene detection (as well as DNA quantitation kits like Helixyte™) to facilitate this. Another approach is immunohistochemistry on tissue sections: using AAT’s PSA™ signal amplification kits, researchers can find an isolated CAR-T cell in a tumor biopsy months after treatment. These methods collectively ensure that, whether the CAR-T cells are abundant or scarce, their long-term presence and distribution can be monitored.

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.

Long-Term Tracking Reagents