GTOnco™ Lymphocyte Activation and Proliferation Assay Service
The lymphocyte activation and proliferation assays are essential and early readout of immune status evaluation and T and B cell functional capacity, which have been serving as critical mechanistic evidence to support and advance immuno-oncology (I-O) drug development. Creative Biolabs offers sophisticated lymphocyte proliferation and cytokine production analysis approaches on our GTOnco™ platform to address these key mechanistic questions in a more physiologically relevant manner than the traditional lymphocyte proliferation assay. By combining highly quantitative flow cytometry and advanced cell-labeling technology, we have established and validated regulatory-compliant lymphocyte proliferation assays to support your I-O drug development efforts.
Lymphocyte Activation and Proliferation Assay Introduction
Lymphocyte activation and proliferation assays (LA&PA) are used for more than just a basic laboratory experiment or a part of a test battery, they have become important early readouts and mechanistic tools to evaluate immune status and T and B cell functional capacity. The data generated from these assays are used as critical mechanistic underpinning evidence for establishing MOAs, screening compounds, biomarker discovery and regulatory submissions.
![Figure 1: Lymphocyte proliferation test—[3H] thymidine uptake. (OA Literature)](static/img/gtonco-lymphocyte-activation-and-proliferation-assay-service-1.jpg)
Figure 1. Lymphocyte proliferation assay—[3H] thymidine incorporation.1
Selection Guides of Lymphocyte Activation and Proliferation Assay
The choice of appropriate detection method depends on the specific research question, sample type, and required resolution.
| Assay Method | Principle | Application & Evidence |
|---|---|---|
| CFSE/CellTrace Dye Dilution | Carboxyfluorescein Succinimidyl Ester (CFSE) or similar dyes bind covalently to intracellular proteins. The dye is halved with each successive cell division. | High-Resolution Kinetics: Flow cytometry analysis of CFSE fluorescence intensity provides a quantitative measure of the division index and precursor frequency, allowing monitoring of up to 8 cell generations. |
| BrdU/3H-Thymidine Incorporation | Deoxynucleoside analogs (BrdU) or radioisotopes (3H) are incorporated into the DNA of dividing cells. | Gold-Standard Quantification: 3H-Thymidine has historically been the gold standard for bulk proliferation rate in the Lymphocyte Transformation Test (LTT). |
| Intracellular Ki-67 Staining | Ki-67 is a nuclear protein present in all active phases of the cell cycle. | Immediate Status Check: Measured by flow cytometry, it provides a snapshot of the percentage of cells that have left the quiescent state. |
| Multiplexed Cytokine Analysis | Measurement of secreted cytokines (e.g., IL-2, IFN-γ, TNF-α) in the culture supernatant via ELISA or bead-based assays. | Functional Output: An indirect measure of activation, as T cell proliferation is often IL-2-dependent. Evidence: The ratio of Th1 (IFN-γ) to Th2 (IL-4) cytokines provides a functional profile of the immune response, critical for characterizing drug-induced immune skewing. |
A Flexible Suite of Services at Creative Biolabs
Cell Proliferation Analysis
- CFSE-based lymphocyte proliferation tracking.
- Multiple fluorochrome options available for different experimental needs.
- Quantitative analysis of cell division generations.
- High-resolution monitoring of proliferation kinetics.
Cytokine Production Assessment
- In vivo cytokine capture assay (IVCCA).
- Detection of activation-induced cytokine release.
- Biotinylated anti-cytokine antibody-based measurements.
- Comprehensive cytokine profile analysis.
Cell Types Analyzed
- T lymphocytes (CD4+ and CD8+ subsets)
- B lymphocytes
- NK cells
- Other specific lymphocyte populations upon request
Analysis Options
- Cell proliferation rate and index.
- Activation marker expression.
- Cytokine production levels.
- Cell viability assessment.
- Population distribution analysis.
Project Execution and Quality Control
Once the experimental protocol is finalized, our team of technical experts will execute the research with meticulous attention to detail and rigorous quality control:
Cell Processing and Culture:
Isolate primary lymphocytes or prepare cell lines under optimized conditions, and closely monitor cell viability and purity.
Experimental Implementation
Precisely execute activation and proliferation protocols, and perform appropriate standardization and controls.
Multi-parameter Data Acquisition
Acquire high-quality data using calibrated instruments and conduct daily quality control checks.
Data Processing and Analysis
Apply advanced analytical workflows to extract meaningful biological information from complex raw data.
Applications of Lymphocyte Activation and Proliferation Assay in Immuno-Oncology
Cancer Immunotherapy Development
Evaluate the effectiveness of cancer immunotherapy treatments by monitoring lymphocyte responses.
- Assessment of immune checkpoint inhibitor efficacy.
- CAR-T cell therapy monitoring.
- Evaluation of cancer vaccine responses.
- Analysis of tumor-infiltrating lymphocyte activity.
Drug Mechanism Studies
Understand the mechanisms of action for immunomodulatory compounds.
- Characterization of immune response patterns.
- Investigation of drug-induced T cell activation.
- Analysis of cytokine production profiles.
- Assessment of immune cell functional changes.
Safety Assessment
Evaluate potential immunological effects and safety profiles of therapeutic candidates.
- Immunotoxicity screening.
- Monitoring of adverse immune responses.
- Assessment of off-target effects.
- Long-term immunity impact evaluation.
Biomarker Development
Identify and validate immune response biomarkers for clinical applications.
- Immune response pattern identification.
- Patient stratification marker discovery.
- Treatment efficacy monitoring.
- Prediction of therapeutic outcomes.
GTOnco™ Platform Advantages and Highlights
Advanced Flow Cytometry Platform
State-of-the-art multicolor flow cytometry systems enable high-resolution analysis of multiple parameters simultaneously with superior sensitivity and precision.
Validated Protocols
Comprehensive validation process ensures reproducibility and reliability. All protocols are optimized according to regulatory guidelines and industry standards.
Customizable Analysis
Flexible experimental design and analysis parameters to meet specific research needs, supported by expert consultation throughout the project.
Data Quality & Security
Rigorous quality control processes and secure data management systems ensure the integrity and confidentiality of your research data.
Rapid Turnaround
Efficient project management and streamlined workflows enable quick delivery of results without compromising quality or accuracy.
Expert Support Team
Experienced scientists provide comprehensive technical support and consultation throughout your project, from design to data interpretation.
Frequently Asked Questions
Q: Can you perform the assay with cryopreserved samples?
A: Yes, we have extensive experience working with cryopreserved samples. However, proper handling and preservation are crucial for successful analysis. Here are our key requirements and recommendations:
-
Viability Requirements
- Minimum post-thaw viability: >90%
- Optimal preservation medium: 90% FBS + 10% DMSO
- Controlled-rate freezing strongly recommended
-
Required Documentation
- Date of sample collection and cryopreservation
- Freezing method and media composition
- Storage conditions and temperature logs
- Any relevant processing steps prior to freezing
-
Quality Control Measures
- Post-thaw recovery and viability assessment
- Functional validation using standard stimulation protocols
- Phenotypic analysis to confirm cell population integrity
Q: What is the typical turnaround time for lymphocyte proliferation assays?
A: Turnaround time varies depending on the type of assay. Standard CFSE-based proliferation assays typically take 5–7 days from cell stimulation to data delivery, as the assay includes initial cell labeling, a 3–5 day culture period, and subsequent analysis. BrdU/EdU incorporation assays can usually be completed within 2–4 days, while metabolic assays (CCK-8/MTS) provide results within 1–3 days. Complex multi-timepoint kinetic studies or studies involving primary cell isolation may extend these times. A detailed timeline based on your experimental design will be provided during the project consultation.
Q: What are the key factors to consider when selecting CFSE, BrdU/EdU, and metabolic assays?
A: CFSE dilution assays are well-suited for tracking multiple cell divisions and assessing proliferative heterogeneity within subsets, providing information about the number of divisions of individual cells. BrdU/EdU incorporation assays can accurately identify the proportion of cells actively synthesizing DNA within a specific time window, making them ideal for synchronization systems or fixed time points. Metabolic assays offer the highest throughput, making them ideal for screening applications, but the proliferation indicators they provide are indirect and susceptible to changes in metabolic activity. Our technical team can help you choose the most suitable method based on your specific research questions and sample limitations.
Q: What sample types and quantities are required for these analyses?
A: We have successfully processed a variety of sample types, including peripheral blood mononuclear cells (PBMCs), isolated T cell subsets, tumor-infiltrating lymphocytes (TILs), lymph node suspensions, and engineered cell therapy products (CAR-T, TCR-T). For standard proliferation analyses using primary human PBMCs, we typically recommend at least 10 to 20 million cells to cover various experimental conditions and controls. For rare cell types or complex multi-arm studies, we can explore strategies to minimize sample requirements through miniaturized analytical methods.
Q: Can these assays be combined with other functional assays?
A: Absolutely. One of the advantages of our method is its ability to combine proliferation assessment with complementary functional markers. Common multiplex assays include:
- Detection of cytokine (IL-2, IFN-γ, TNF-α, etc.) production via intracellular staining or supernatant analysis;
- Surface phenotypic analysis of activation markers (CD25, CD69, CD71) and differentiation antigens (CD45RA, CCR7, CD62L);
- Expression of memory cell subset differentiation and exhaustion markers (PD-1, TIM-3, LAG-3);
- Assessment of cytotoxic potential via granzyme B, perforin, or CD107a degranulation assays.
Connect with Us Anytime!
Creative Biolabs remains at the forefront of lymphocyte functional analysis, continuously refining and expanding its service portfolio to address emerging challenges in immunology and immunotherapy development. Our comprehensive lymphocyte activation and proliferation assays provide researchers with reliable and meaningful data, facilitating the smooth progression of their projects from fundamental discovery to translational development. Contact us for free expert advice to support all of your I-O assay needs.
Reference
- Ganesan N, Ronsmans S, Hoet P. Methods to assess proliferation of stimulated human lymphocytes in vitro: a narrative review. Cells, 2023, 12(3): 386. https://doi.org/10.3390/cells12030386 (Distributed under Open Access license CC BY 4.0, without modification.)
