Ki-67 is an intranuclear protein selectively expressed in proliferating cells throughout the cell cycle, but absent in resting cells (G0). This expression pattern has made Ki-67 a standard marker for quantifying and identifying actively proliferating cells by flow cytometry and other applications. In flow cytometry, cells are first fixed and permeabilized, then stained with fluorochrome-conjugated anti–Ki-67 antibodies. Ki-67 is commonly used in multicolor panels to assess proliferation within phenotypically and functionally defined cell populations, making it a valuable marker for studying cellular dynamics across a broad range of immunological and translational research applications, including immune monitoring, neoplasm profiling, drug development, and investigations of mechanism of action.
Ki-67, a Standard for Identifying Cell Proliferation
- T cell proliferation and immune activation
Understanding immune activation in response to antigens, infection, or immunomodulatory interventions requires measuring proliferative activity within defined T-cell populations. Ki-67 is a widely used marker of T-cell proliferation that can be incorporated into multicolor panels for subset analysis, including assessment of proliferating regulatory T cells through Ki-67/FoxP3 co-staining.
- Tumor proliferation index and oncology research
The proliferative activity of malignant cells is a useful indicator of aggressiveness and treatment response. Combining Ki-67 and phenotypic marker detection by flow cytometry allows quantification of the proliferating fraction of neoplastic cells directly in donor-derived samples and tumor-infiltrating lymphocytes. In drug discovery and translational research, this information helps characterize tumor responses to targeted therapies and identify potential biomarkers.
- CAR-T cell manufacturing and quality control
Monitoring CAR-T-cell expansion during manufacturing goes beyond viability to confirm that CAR-positive cells are actively cycling. Ki-67 can be incorporated into multiparameter flow cytometry panels together with CAR detection, CD4, CD8, viability, and differentiation markers. When combined with functional markers (such as Granzyme B and IFN-γ), Ki-67 also allows coupling proliferative information with effector phenotypes during development and product characterization.
- Vaccine research and infectious diseases
After vaccination or infection, antigen-specific T cells may be rare in blood samples, and their proliferative response can be brief. Ki-67 expression is exclusive to proliferative cells, which makes it a sensitive marker for detecting low-frequency proliferating populations and capturing early immune responses. Because Ki-67 captures recent or ongoing cell-cycle activity without requiring ex vivo stimulation, it is well suited for longitudinal studies, enabling researchers to track changes in the proliferative activity of immune cells over time.
Getting Started with Ki-67 Staining
When setting up a Ki-67 assay, there are a few factors to consider, all related to its intranuclear localization: proper permeabilization and coordination of co-staining with fixation steps.
- Fix and permeabilize for nuclear access
BD Pharmingen™ Transcription Factor Buffer Set is the preferred option for multicolor panels that combine Ki-67 with nuclear transcription factors such as FoxP3. Other kits, including the BD IntraSure™ Kit, have been independently validated for specific applications, such as Ki-67 staining with clone B56 in B-cell neoplasms. Ki-67/FoxP3 co-staining protocols with BD Pharmingen™ Human FoxP3 Buffer set or BD Pharmingen™ Mouse FoxP3 Buffer set are also available. The BD Cytofix/Cytoperm™ Kit is not recommended for Ki-67 staining. Its saponin-based permeabilization does not provide reliable access to nuclear targets, despite its use in some published studies.
- Stain surface markers and viability before fixation
For Ki-67 co-staining workflows, surface markers are typically stained before fixation and permeabilization to preserve antigen detection, as some reagents may alter antibody-binding epitopes. These methods can also affect protein-based fluorochromes such as PE, APC, and their tandems. For improved signal stability, consider fixation-resistant fluorochromes such as BD Horizon™ RealDyes™. If viability assessment is required, use a BD Horizon™ Fixable Viability Stain, as non-fixable dyes such as 7-AAD and propidium iodide (PI) are not retained during intracellular staining workflows. Following fixation and permeabilization, Ki-67 and other intracellular markers, such as FoxP3 and Granzyme B[1] , can be stained
- Find the Ki-67 antibody that matches your needs
Our validated Ki-67 antibody portfolio for RUO flow cytometry covers two clones and multiple fluorochrome conjugates for intranuclear detection in human and mouse samples. Explore our portfolio below and confirm species reactivity and protocol compatibility.
| Clone | Species Reactivity | Isotype | Research Application |
|---|---|---|---|
| B56 | Human (QC Testing), Mouse (Tested in Development), Others* (Reported) | Mouse IgG1, κ | Intracellular staining (flow cytometry) |
| Ki-67 | Human (QC Testing) | Mouse BALB/c IgG1, κ | Intracellular staining (flow cytometry) |
*Clone B56 has reported reactivity (literature) to Rat, Rhesus, Cynomolgus, Baboon, Dog, and Chicken.
Design Multicolor Ki-67 Panels with Confidence
Quantification and identification of proliferative cells within defined populations is typically achieved by integrating Ki-67 into multicolor panels. The choice of markers depends on the application and scientific question to answer.
The most common combinations are used in immune flow panels to assess proliferative subpopulations of T-cell lineages (CD3, CD4, and CD8), characterize Tregs (FoxP3), measure T-cell activation (CD69), or evaluate cytolytic effector potential (Granzyme B*). Additional combinations are used for CAR-T panels when checking for expansion potential (surface and viability markers, including CD4 and CD8), and for oncology panels, where Ki-67 is coupled with neoplastic-relevant markers to calculate the proliferation index and estimate malignancy aggressiveness.
Successful multicolor panel design begins with careful selection of fluorochrome combinations. With our Spectrum Viewer and panel design resources, you can compare fluorochrome spectra, evaluate potential spillover, and assign dyes to your markers based on your specific instrument configuration.
*Important note: Granzyme B is an intracytoplasmic target. Carefully select the fix/perm protocol to match both intranuclear and intracytoplasmic targets.
Build your Ki-67 staining panel
- BD Pharmingen™ Transcription Factor Buffer Set.
Provides the fixation and permeabilization workflow needed for Ki-67 co-staining with FoxP3 and other nuclear targets.
- BD Horizon™ Fixable Viability Stains.
Crucial for viability assessment throughout the Ki-67 staining workflow and should be added before fixation. Multiple options for different laser configurations.
- CD3, CD4 and FoxP3 Antibodies.
A common co-staining antibody used in Ki-67 panels, allowing identification of proliferating regulatory T cells using the same nuclear staining workflow.
- CD19 and CD20 Antibodies.
Used together with Ki-67 and other specific markers to research proliferation of B-cell neoplasm.
- CD3 and CD8 Antibodies.
Used together with Ki-67 to identify and quantify proliferating CD8+ T-cell populations. Surface staining should be performed before fixation.
- BD Horizon™ RealDyes.
A family of fluorochromes for improved signals that is fully compatible with fixation and permeabilization protocols. Suited for both surface and intracellular targets.
FAQs
- What is Ki-67 and why is it used as a proliferation marker?
Ki-67 is an endogenous nuclear protein expressed during the active phases of the cell cycle (G1, S, G2, and M) and absent from resting cells in G0. This expression pattern provides a clear distinction between proliferating and non-proliferating populations. As a result, Ki-67 is widely used as a proliferation marker in T-cell activation, tumor and cancer profiling, CAR-T manufacturing, vaccine immunogenicity, and other flow cytometry research applications.
- What fixation and permeabilization protocol is required for Ki-67 intranuclear staining?
BD Pharmingen™ Transcription Factor Buffer Set, which is preferred for panels combining Ki-67 with other nuclear targets such as FoxP3. BD Cytofix/Cytoperm™ is not recommended for Ki-67 staining because its saponin-based permeabilization may not reliably provide access to nuclear targets.
- Can I co-stain Ki-67 and FoxP3 in the same panel?
Yes, Ki-67 and FoxP3 can be co-stained using the BD Pharmingen™ Transcription Factor Buffer Set, which provides access to both nuclear targets. For multicolor panels, stain surface markers such as CD4, CD8, or CD25 and add the viability dye before fixation. After nuclear permeabilization, FoxP3 and Ki-67 can be stained simultaneously. Validated protocols are available for human PBMCs and mouse splenocytes.
- What are common markers to combine with Ki-67 in a flow cytometry panel?
Ki-67 is commonly combined with lineage markers (CD3, CD4, CD8, and FoxP3), activation markers (CD25 and CD69), and effector markers (Granzyme B) to assess T-cell subsets, activation, effector function, and Treg populations. Other combinations include those for determining CAR-T expansion (CD4, CD8 and viability markers) and for calculating proliferation index in oncology research panels (EpCAM and cytokeratins, among others).
- What is the difference between Ki-67 and other proliferation markers like BrdU?
Ki-67 is an endogenous nuclear marker that reflects active cell-cycle status at the time of staining, without requiring exogenous labeling or cell culture manipulation. BrdU is an exogenous nucleotide analog incorporated into newly synthesized DNA, providing a specific readout of S-phase activity after in vivo or in vitro administration. Ki-67 is therefore useful to assess proliferation with minimal sample manipulation, while BrdU can be valuable for specifically measuring DNA synthesis or tracking proliferation over time in research applications.