As part of adaptive immunity, T cells play an integral role in executing and controlling immune responses. T cells can be distinguished from other lymphocytes, such as B cells and natural killer (NK) cells, by the presence of a T cell receptor on the cell surface. Each of the various T cell subsets has distinct functions and aids the immune response to achieve best efficacy.
Maturing from thymocytes, T cells undergo several development stages: After selection in the thymus, they circulate the body as naive T cells, each with a unique antigen specificity. Upon antigen encounter, they are activated, differentiate into a specific subtype, expand, and fulfill their role as effector cells, e.g., by migrating into various tissues and organs. For long-lasting immune memory, some of the antigen-activated T cells differentiate into various memory T cell subtypes.Blood (human)
At a glance: T cell subsets in peripheral blood (broad classification)
Cell type or subset | Frequency | Markers | Function |
T cells (overall) | 15–30% of CD45+ leukocytes in peripheral blood | CD45, CD3, CD2, CD5, CD6 | Regulators and effectors of the adaptive immune response |
CD4+ T helper cells | 9–20% of CD45+ leukocytes in peripheral blood | CD45, CD3, CD4 |
|
CD4+CD25+ regulatory T cells | <1% of CD45+ leukocytes in peripheral blood | CD45, CD3, CD4, CD25 |
|
CD8+ cytotoxic T cells | 5–10% of CD45+ leukocytes in peripheral blood | CD3, CD8 |
|
TCRγ/δ+ T cells | <10% of all T cells in peripheral blood | γ/δ TCR+, CD3, CD4+/–, CD8+/– |
|
Natural killer T cells (NKT cells) | <3% of all T cells in peripheral blood | CD45, CD3, CD56, CD16, NK1.1, granzyme |
|
Double-negative T cells | 1–3% of all peripheral T cells | α/β TCR+, CD3 |
|
The range of functionally diverse T cell subtypes can be divided broadly into CD4+ T helper cells, CD8+ cytotoxic T cells, CD4+CD25+FoxP3+ regulatory T cells, and the 'unconventional' T cell subtypes, such as γ/δ T cells, NKT cells, and double-negative T cells. Thus, T cells are typically identified by their expression of CD3 together with either CD4 or CD8. Additional markers are used to further distinguish subtypes.
Most T cell subtypes can undergo memory differentiation steps after activation by their respective antigen. Apart from differentiating into effector T cells, some naive T cells (TNaive) may differentiate into various memory T cells subsets, such as stem cell–like memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM) and effector memory RA+ T cells (TEMRA). Each differentiated subset is defined by distinct surface markers. Antigen-inexperienced T cells express the naive marker CD45RA, as well as homing receptors CD62L and CCR7, but lack CD45RO and CD95 expression. With ongoing differentiation towards memory phenotypes, CD45RA, CD62L, and CCR7 are down-regulated, while memory marker CD45RO and activation marker CD95 are gradually up-regulated. With progressive differentiation towards the memory phenotype, antigen dependence, tissue tropism, effector function, and senescence increase (PMID: 24258910, 26999211).
Shared features of all memory T cell subtypes is that they are long-lived and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thereby mounting a faster and more potent immune response than the first immune response to a given pathogen. The different subtypes exert different functions and exhibit different properties, such as tissue tropism or capacity for self-renewal, reflecting the specific immune-related circumstances that led to their differentiation into a given memory subtype.Typically, the frequency of naive T cells specific for a given antigen is very low, ranging between 0.01 and 0.001% of the total T cell count, depending on the respective specificity. When a naive T cell encounters its cognate antigen and is consequently activated, clonal expansion begins, boosting the frequency of those antigen-specific T cells by several orders of magnitude. This way, they can efficiently fulfill their role as effectors in the immune response (PMID: 22517866, 17707129).
Most clonally expanded antigen-specific T cells die after the termination of the immune response, but a small percentage survive as memory T cells. Memory T cells have a long lifespan and can quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen. At birth, the T cell repertoire is almost exclusively composed of naive T cells. With progressing age and antigen experience, memory T cells may become the most abundant T cell population, constituting up to 35% of all circulating T cells (PMID: 24336101).
Notably, laboratory mice carry almost exclusively naive T cells due to their specific holding conditions and relatively young average age. This, of course, changes dramatically in certain disease-related experimental settings. In humans, the frequency of naive and memory T cells greatly depends on age, living conditions, and individual history of immune responses.Miltenyi Biotec has created dedicated application protocols for the isolation, activation, and expansion of pan T cells.
Blood (human)
Miltenyi Biotec has developed numerous products for the magnetic separation of T cells from whole blood and from PBMCs. For details on MACS® Cell Separation Technology, see the handbook chapter Magnetic cell separation.
The following is a description of Miltenyi Biotec solutions for the isolation of pan T cells, including activated, naive or memory T cells, TCRγ/δ+ T cells, and NKT cells. Other chapters describe dedicated solutions for CD4+ T helper cells, regulatory T cells, and CD8+ cytotoxic T cells.Magnetic cell separation
At a glance: Kits and reagents for the separation of pan T cells from whole blood, buffy coat, and LRSC
Starting material | Isolation strategy | Comments | Automation | Product |
Whole blood | Depletion of non-target cells | Isolation of pan T cells directly from up to 30 mL whole blood in less than 30 minutes. | No | MACSxpress® Whole Blood Pan T Cell Isolation Kit, human |
Buffy coat | Depletion of non-target cells | Isolation of pan T cells directly from an entire buffy coat in less than 30 minutes. | No | MACSxpress® Buffy Coat Pan T Cell Isolation Kit, human |
LRSC | Depletion of non-target cells | Isolation of pan T cells directly from LRSC in less than 30 minutes. | No | MACSxpress® LRSC Pan T Cell Isolation Kit, human |
Whole blood | Positive selection of target cells | Isolation of Pan T cells directly from whole blood. | Yes* | StraightFrom® Whole Blood CD3 MicroBeads, human |
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. |
The StraightFrom® Whole Blood CD3 MicroBeads, human, were developed for the rapid positive selection of CD3+ T cells directly from whole blood. The kit requires no sample preparation, like density gradient centrifugation, erythrocyte lysis, or cell count.
Highly standardized and fast isolation of CD3+ cells from whole blood. Isolation of CD3+ cells from whole blood using StraightFrom Whole Blood CD3 MicroBeads and the MultiMACS™ Cell24 Separator Plus with Single-Column Adapter and Whole Blood Columns. Cells were fluorescently stained with CD3-PE, CD14‑APC, and CD45-VioBlue® for flow cytometry analysis on the MACSQuant® Analyzer. Cells were triggered via CD45-VioBlue. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
Untouched Pan T cells isolated from 30 mL of human EDTA-anticoagulated whole blood. Samples were processed using the MACSxpress Whole Blood Pan T Cell Isolation Kit, a MACSmix™ Tube Rotator, and a MACSxpress Separator. The isolated cells were fluorescently stained with CD45-VioBlue, CD3-FITC, CD3‑APC, CD2-PE, and CD56-PE and then analyzed by flow cytometry on the MACSQuant Analyzer. Cell debris, non-leukocytes, and dead cells were excluded from the analysis based on CD45 expression, scatter signals, and propidium iodide fluorescence.
MACS Cell Separation - Select the best (brochure)
StraightFrom MicroBeads (brochure)
At a glance: Kits and reagents for the separation of Pan T cells from PBMCs
Starting material | Isolation strategy | Comments | Automation | Product |
PBMCs | Positive selection of target cells | CD2 is one of the earliest T cell markers, involved in thymic development. | Yes* | CD2 MicroBeads, human |
PBMCs | Positive selection of target cells | CD3 is expressed on all T cells and the most reliable pan T cell marker. Ideal for depletion of T cells from mixed cell suspensions. | Yes* | CD3 MicroBeads, human |
PBMCs | Positive selection of target cells and subsequent label removal | The kit allows the isolation of label-free CD3+ cells, because the complete labeling complex can be released from the cell surface after separation. | No | REAlease CD3 MicroBead Kit, human |
PBMCs | Positive selection of target cells | CD6 is expressed on the majority of T cells, as well as on a subset of B cells. | Yes* | CD6 MicroBeads, human |
PBMCs | Depletion of non-target cells | Isolation of highly pure T cells is achieved by depletion of non-target cells: monocytes, neutrophils, eosinophils, B cells, stem cells, dendritic cells, NK cells, granulocytes, and erythroid cells. | Yes* | Pan T Cell Isolation Kit, human |
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. |
Instead of working directly with whole blood, samples can be processed by density gradient centrifugation to pre-enrich peripheral blood mononuclear cells (PBMCs) as starting material for subsequent T cell isolation.
The Pan T Cell Isolation Kit, human was developed for fast isolation of untouched T cells from PBMCs in only 18 minutes. Non-T cells (i.e., monocytes, neutrophils, eosinophils, B cells, stem cells, dendritic cells, NK cells, granulocytes, and erythroid cells) are labeled with a cocktail of biotin-conjugated antibodies against CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a (Glycophorin A). Subsequently, these non-target cells are magnetically labeled with the Pan T Cell MicroBead Cocktail, and isolation of highly pure T cells is achieved by depletion of the magnetically labeled cells.Untouched T cells isolated from PBMCs. Non-T cells were depleted using the Pan T Cell Isolation Kit, an LS Column, and a MidiMACS™ Separator. Cells were labeled with CD2-PE and CD3-FITC and then analyzed by flow cytometry on the MACSQuant Analyzer.
MACS Cell Separation (brochure)
At a glance: Kits and reagents for the separation of naive/memory T cell subsets from PBMCs
Starting material | Isolation strategy | Comments | Automation | Product |
PBMCs | Depletion of non-target cells | Depletion of memory T cells and non-T cells, such as B cells, NK cells, macrophages, stem cells, platelets, granulocytes, and monocytes. Includes optional depletion of TCRγ/δ+ T cells. | Yes* | Naive Pan T Cell Isolation Kit, human |
PBMCs | Positive selection of target cells | Isolation of naive T cells, both CD4+ and CD8+. CD62L is expressed on all lymphocytes, not only T cells. | Yes* | CD62L MicroBeads, human |
PBMCs | Positive selection of target cells | Isolation of naive T cells, both CD4+ and CD8+. CD45RA is also expressed on CD8+ TEMRA cells. CD45RA expression is not limited to T cells, but it can also be expressed by other lymphocytes. | Yes* | CD45RA MicroBeads, human |
PBMCs | Positive selection of target cells | Isolation or depletion of memory T cells, both CD4+ and CD8+. CD45RO is not expressed on CD8+ TEMRA cells. CD45RO expression is not limited to T cells, but it can also be expressed by other lymphocytes. | Yes* | CD45RO MicroBeads, human |
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. |
Efficient isolation of untouched naive T cells by depletion of non-T cells. Separation was done from PBMCs using the Naive Pan T Cell Isolation Kit, an LS Column, and a MidiMACS Separator. Cells were labeled with CD3-VioBlue, CD197-PE, and CD45RA-FITC, and then analyzed by flow cytometry on the MACSQuant Analyzer.
The CD62L MicroBeads, CD45RA MicroBeads, or CD45RO MicroBeads are used to positively select the respective cells according to the given marker. Isolation of CD62L+, CD45RA+, or CD45RO+ T cells can be accomplished by using these MicroBeads in combination with kits enabling the isolation of untouched T cells, like the Pan T Cell Isolation Kit.
At a glance: Kits and reagents for the separation of activated T cells from PBMCs
Starting material | Isolation strategy | Comments | Automation | Product |
PBMCs | Positive selection of target cells | CD137 is involved in the activation and survival of CD4+ and CD8+ T cells, and NK cells. | Yes* | CD137 MicroBead Kit, human |
PBMCs | Positive selection of target cells | CD154 is up-regulated on activated CD4+ T cells. | Yes* | CD154 MicroBead Kit, human |
PBMCs | Positive selection of target cells | CD25, the low-affinity interleukin-2 receptor alpha chain (IL-2Rα), is expressed on activated T and B cells and monocytes. CD25 is also present on a subset of non-activated CD4+ T cells, which has regulatory function (Treg cells). | Yes* | CD25 MicroBeads II, human |
PBMCs | Positive selection of target cells | CD30 is expressed on small subsets of activated T and B cells, but it is not found on resting lymphocytes or monocytes. | Yes* | CD30 MicroBeads, human |
PBMCs | Positive selection of target cells | CD69 is expressed on activated T cells, B cells, and NK cells, but not on resting lymphocytes. It is rapidly up-regulated after lymphocyte activation and also expressed on activated macrophages and other cell types, including neutrophils, eosinophils, and platelets. | Yes* | CD69 MicroBead Kit II, human |
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. |
T cell activation can be i) antigen specific, by recognition of the cognate antigen, or ii) polyclonal via, e.g., antibody-mediated CD3/CD28 ligation, binding of chemical agents like PMA/ionomycin, or exposure to a super-antigen like staphylococcal enterotoxin B (SEB) or the non-toxic alternative CytoStim™. T cell activation is characterized by up-regulation of surface markers and the expression of proinflammatory cytokines. Depending on the T cell subtype and the activation mode, activation markers and cytokines include CD25 (IL2RA), CD30, CD38, CD69, CD95 (FASR), CD137 (4-1BB), CD154 (CD40L) and IL-2, IL-4, IL-5, IL-6, IL-9, IL-13, IL17, TNF-α, IFN-γ, respectively. For details on using these markers to identify activated T cells, see Characterization of T cells by flow cytometry below.
Antigen-activated T cells are especially interesting for research because of their specificity and potential for cell-based therapies. However, the frequency of antigen-specific T cells in peripheral blood can be as low as 0.01% of all T cells. This low frequency makes processing of high cell numbers and the use of specialized analysis methods essential for reliable detection and phenotypic characterization. Miltenyi Biotec has developed dedicated application protocols for working with antigen-specific T cells.
CD137 and CD154 were described as surrogate markers for antigen-activated T cells and can be used for analysis and enrichment of these rare cells. CD137 is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells, and on natural killer cells, but can also be found on resting monocytes and dendritic cells. CD137 has been described as a suitable marker for antigen-specific activation of human CD8+ T cells, as CD137 is not expressed on resting CD8+ T cells and its expression is reliably induced after 24 hours of stimulation. Thus, activated antigen-specific CD8+ T cells can be isolated by combining the CD137 MicroBead Kit, human with the CD8+ T Cell Isolation Kit, human.
The CD154 antibody specifically recognizes the human CD154 antigen, also known as CD40L, gp39, T-BAM, TRAP, or Ly-62. CD154 is transiently up-regulated on activated CD4+ T cells and plays an important role as a costimulatory molecule in T cell/antigen-presenting cell interactions through ligation of CD40. Due to the transient expression of CD154 within hours after activation, the CD154 MicroBead Kit, human can be used to isolate activated antigen-specific CD4+ T cells.
Secreted cytokines can be used either as markers for antigen-activated T cells, or as a means to isolate and analyze these cells. For details, see Analysis of surface markers and cytokines below. Miltenyi Biotec has also created dedicated application protocols for working with cytokine-secreting cells.
Characterization of antigen-specific naive and memory T cell subsets (Application note)
At a glance: Kits and reagents for the separation of activated T cells from PBMCs
Starting material | Isolation strategy | Comments | Automation | Product |
PBMCs | Positive selection of target cells | Direct immunomagnetic separation via the γ/δ variant of the T cell receptor (TCRγ/δ). | Yes* | Anti-TCRγ/δ MicroBead Kit, human |
PBMCs | Depletion of non-target cells | Non-TCRγ/δ+ T cells (TCRα/β+ T cells, NK cells, B cells, dendritic cells, granulocytes, monocytes, stem cells, and erythroid cells) are indirectly labeled and depleted by immunomagnetic separation. | Yes* | TCRγ/δ+ T Cell Isolation Kit, human |
PBMCs | Positive selection of target cells | Positive selection of human invariant natural killer T cells (iNKT cells) based on the expression of the TCR α-chain Vα24-Jα18. | Yes* | Anti-iNKT MicroBeads, human |
PBMCs | Depletion of non-target cells | NK cells and monocytes are first magnetically labeled and depleted. CD3+CD56+ NKT cells are then positively selected from the pre-enriched NKT cell fraction using CD56 MicroBeads. | Yes* | CD3+CD56+ NKT Cell Isolation Kit, human |
PBMCs | Depletion of non-target cells followed by positive selection of target cells | CD4–CD8–CD56–CD3+TCRα/β+ cells are isolated from PBMCs in a two-step procedure. | (Yes*) -two steps | Double-negative T Cell Isolation Kit, human |
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. |
Gamma/delta T cells (γ/δ T cells) have a distinctive T cell receptor (TCR) on their surface that consists of one γ chain and one δ chain, as opposed to the 'conventional' α/β T cells that express a TCR composed of α and β TCR chains. Since most T cells are α/β T cells, γ/δ T cells are relatively rare (<10% of all T cells in peripheral blood), showing highest abundance in a population of lymphocytes of the gut mucosa known as intraepithelial lymphocytes.
The antigens that activate γ/δ T cells and their specific function are still largely unknown. They recognize antigens without the presence of major histocompatibility complex molecules, are able to function as antigen-presenting cells, and they exhibit cytotoxicity. This makes them ideal candidates as therapeutic targets to induce durable immunity in the context of different pathologies. Their functional responses are induced upon recognition of stress antigens, such as heat-shock proteins, which promotes cytokine production and regulates various stages of the immune reaction in response to stress. Thereby, γ/δ T cells can attack target cells directly through their cytotoxic activity or indirectly through the activation of other immune cells.
The TCRγ/δ+ T Cell Isolation Kit, human, was developed to isolate untouched human γ/δ T cells from PBMCs. Non-TCRγ/δ+ cells are indirectly magnetically labeled with a cocktail of biotinylated monoclonal antibodies and Anti-Biotin MicroBeads. The magnetically labeled non-TCRγ/δ+ cells are retained on the column in the magnetic field of a MACS Separator, while the unlabeled γ/δ+ T cells pass through.Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties with both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids. As such, NKT cells are important in recognizing glycolipids from organisms, such as Mycobacterium tuberculosis.
NKT cells express an α/β T cell receptor, but also a variety of molecular markers that are typically associated with NK cells, such as NK1.1. The best-known NKT cells differ from conventional α/β T cells in that their T cell receptors are far more limited in diversity ('invariant' or 'type 1' NKT cells, also termed iNKT cells). However, also other CD1d-restricted T cells ('type 2' NKT cells) recognize lipids and glycolipids presented by CD1d molecules, rather than peptide-MHC complexes. NKT cells include both NK1.1+ and NK1.1−, as well as CD4+, CD4−, CD8+ and CD8− cells. NKT cells may share other features with NK cells, such as CD16 and CD56 expression and granzyme production.
In humans, iNKT cells express a highly conserved TCR, consisting of Va24-Ja18 paired with Vb11, which is specific for glycolipid antigens. They are notable for their ability to respond rapidly to danger signals and pro-inflammatory cytokines. Once activated, they engage in effector functions, like NK transactivation, T cell activation and differentiation, B cell activation, dendritic cell activation and cross-presentation activity, and macrophage activation.
The CD3+CD56+ NKT Cell Isolation Kit, human, was developed for the sequential separation of CD3+CD56+ NKT cells from human PBMCs and other single-cell suspensions.
The down-regulation of immune responses by regulatory T cells plays a key role in the induction of tolerance. It has been reported that TCRα/β+CD4–CD8– double-negative T cells also have the ability to specifically down-regulate immune responses towards allo-antigens, both in humans and mice. Double-negative T cells are found in lymphoid as well as in non-lymphoid tissues. They constitute about 1–3% of peripheral CD3+ cells.
The Double-negative T Cell Isolation Kit enables the isolation of double-negative T cells (CD4–CD8–CD56–CD3+TCRα/β+ cells) from PBMCs by sequential sorting. The isolation is performed in a two-step procedure.
Isolation of double-negative T cells. TCRα/β+CD4–CD8– double-negative T cells were isolated from human PBMCs using the Double-negative T Cell Isolation Kit, one LD Column, two MS Columns, a MidiMACS Separator, and a MiniMACS Separator. Cells were also fluorescently stained with Anti-Biotin-FITC.
T cells can be detected by flow cytometry based on their expression of cell surface markers, transcription factors, and the cytokines they produce. Depending on the subpopulation of interest, a specific combination of markers can be used for characterization. Miltenyi Biotec offers a vast portfolio of conventional and recombinant REAfinity™ antibodies and flow analysis kits for comprehensive analysis.
The following surface markers, cytokines, and transcription factors can be used to identify T cells and subsets according to their development state or subtype.
At a glance: Markers for the detection of T cells by flow cytometry
T cell development – naive vs. memory (TSCM/TCM/TTM/TEM/TEMRA) | Activated T cells | Exhausted T cells | TCRγ/δ+ T cells | NKT cells |
---|---|---|---|---|
CD3 | CD3 | CD3 | TCR γ/δ | CD3 |
CD4 | CD4 | CD4 | CD3 | CD4+/– |
CD8 | CD8 | CD8 | CD4+/– | CD8+/– |
CD27 | CD25 (IL2RA) | CD96 (TACTILE) | CD8+/– | CD16 |
CD28 | CD69 | CD152 (CTLA-4) | CD56 | |
CD45RA | CD95 (FasR) | CD223 (LAG-3) | CD57 | |
CD45RO | CD134 (OX40) | CD244 (2B4) | CD161 (NK1.1) | |
CD57 | CD137 (4-1BB) | CD272 (BTLA) | TCR Vα24-Jα18 with TCR Vβ11 (iNKT) | |
CD62L (L-Selectin) | CD154 (CD40L) | CD278 (ICOS) | ||
CD69 | Ki-67 | CD279 (PD1) | ||
CD95 (FasR) | KLRG1 | CD366 (TIM-3) | ||
CD127 | TIGIT | |||
CD197 (CCR7) | VISTA | |||
EOMES |
REAfinity Recombinant Antibodies (brochure)
Recombinant antibodies for improved standardization in flow cytometry (scientific poster)
Miltenyi Biotec offers a range of solutions for the analysis of T cell–associated surface markers and cytokines:
Miltenyi Biotec offers a specialized and versatile range of culture media and reagents for the stimulation, activation/expansion, and differentiation of T cells.
At a glance: Kits and reagents for the cultivation, activation, and expansion of T helper cells
Use | Comments | Product |
Culture medium | Optimized T cell media without serum or animal-derived components. Also available in MACS GMP grade and with or without phenol red. | TexMACS Medium |
Supplement | Consistent, high-quality recombinant cytokines for successful cell culture. Available in premium, research and MACS GMP grades. | MACS Cytokines |
Stimulation | Nanomatrix-based activation of T cells via CD3/CD28 engagement.Available in research, and MACS GMP grades. | T Cell TransAct |
Stimulation | Cell-sized activation beads (‘artificial antigen-presenting cells’) loaded with activating CD2, CD3, and CD28 antibodies. | T Cell Activation/Expansion Kit, human |
Stimulation | Non-toxic alternative to Staphylococcal enterotoxin B (SEB). Functions as a superantigen. | CytoStim |
Stimulation | Extensive panels of tumor-, virus-, fungi- and microbiota-specific antigens for the stimulation of antigen-specific CD4+ and CD8+ T cells. Available in premium, research, and MACS GMP grades as well as in 96-well cell culture plate format. | PepTivator Peptide Pools |
Stimulation | In vitro T cell activation and expansion. | CD28 pure – functional grade, human |
TexMACS™ Medium is a serum-free cell culture medium developed specifically for T cells. It has been used in a variety of applications and, in combination with MACS Cytokines, is an ideal starting point for reliable cultivation conditions. The medium is also available in MACS GMP grade, and with or without phenol red.
For detailed information about Miltenyi Biotec media optimized for T cells, see the MACS Handbook chapter Cell culture.
T cell activation is essential for a variety of downstream application. Miltenyi Biotec offers polyclonal stimulation reagents that have been carefully designed to ensure optimal stimulation conditions.
T Cell TransAct™ is a ready-to-use reagent that is applied volumetrically, eliminating the need for bead-to-cell ratio calculations. Excess reagent is simply removed via culture wash. T Cell TransAct is available in both research and MACS GMP grades for a seamless transfer of workflows into clinical settings.
The T Cell Activation/Expansion Kit, human, employs large cell-sized particles loaded with biotinylated antibodies of choice to activate and expand primary cells. The large cell-sized particles mimic antigen-presenting cells and, when loaded with CD2, CD3, and CD28 antibodies and applied in a specific bead-to-cell ratio, lead to efficient T cell activation. The decision tree below helps to find the right T cell activation product for a particular experiment.
CytoStim™ is an antibody-based reagent that rapidly stimulates T cells. It can be used as a non-toxic alternative to SEB, e.g., for the positive control of antigen-specific T cell stimulation assays or intracellular cytokine staining experiments to detect cytokine or activation marker expression.
PepTivator® Peptide Pools enable the antigen-specific stimulation of both CD4+ and CD8+ T cells with an extensive panel of tumor-, virus-, fungi- and microbiota-specific antigens. Consisting of 15-mer peptides with 11-amino-acid overlaps, PepTivator Peptide Pools cover the complete sequence of the respective antigen. Available in research-, premium- and MACS GMP grades. The most popular PepTivator Peptide Pools are also available in a 96-well cell culture plate format for high-throughput cell activation.
MACS Cytokines are available in three different grades – research, premium, and MACS GMP grades – to provide best flexibility in any assay setup. Notably, premium-grade MACS Cytokines exhibit well-defined biological activities, normalized to international reference standards (IU/mg), that allow exact unit dosing for reproducible results without laborious pre-testing.
Finally, the CD3 and CD28 pure – functional grade antibodies, human are suitable for in vitro T cell activation and expansion. The CD3 (OKT3) andCD28 (15E8) antibodies recognize the respective human receptors. Upon receptor binding, a stimulatory signal is transferred that, in combination with additional cytokines (e.g., IL-2 or IL-7/IL-15), leads to the activation and expansion of T cells.Cell culture
PepTivator Peptide Pools (brochure)
T Cell TransAct (brochure)
Miltenyi Biotec has created dedicated application protocols to enrich T cells from various human tissues:
Tissues must be dissociated into a single-cell suspension for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis. Combining mechanical dissociation and enzymatic treatment by using the gentleMACS™ Dissociator with Heaters and specific Tissue Dissociation Kits enables reproducible T cell isolation. T cells can be obtained with excellent yield, high viability rate, and preserved cell epitopes even from hard-to-process tissues, including tumor, skin, and brain tissue, among others. See Human cells and organs to learn more about the sample preparation of different tissue types.
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