MACS Handbook

Human Pan T cells

1 Introduction

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.

2 T cells from peripheral blood

In human peripheral blood, 15–30% of all CD45+ leukocytes are T cells, with the CD4+ T cells accounting for approximately two thirds of the total T cell population, and CD8+ T cells making up the remaining one third. In isolated PBMCs, T cells are with 45–70% of total cells by far the most abundant cell type. Up to 60% of total PBMCs are CD4+ T cells and up to 30% are CD8+ T cells.
MACS Handbook:

Blood (human)

2.1 Cell subsets, frequencies, marker expression

At a glance: T cell subsets in peripheral blood (broad classification)

Cell type or subsetFrequencyMarkersFunction
T cells (overall)15–30% of CD45+ leukocytes in peripheral bloodCD45, CD3, CD2, CD5, CD6Regulators and effectors of the adaptive immune response
CD4+ T helper cells9–20% of CD45+ leukocytes in peripheral bloodCD45, CD3, CD4
  • Regulators of adaptive immune response
  • Secrete cytokines to direct immunologic processes
  • Stimulate maturation of B cells
  • Activate cytotoxic T cells and macrophages
  • Differentiation into various subsets (e.g., TH1, TH2, TH17)

CD4+CD25+ regulatory T cells


<1% of CD45+ leukocytes in peripheral bloodCD45, CD3, CD4, CD25
  • Terminate the immune response
  • Suppress autoreactive T cells
  • Maintain immunological tolerance
  • FoxP3 as defining transcription factor
CD8+ cytotoxic T cells5–10% of CD45+ leukocytes in peripheral bloodCD3, CD8
  • Executors of the cellular immune response
  • Antigen-specific killing of, e.g., virus-infected and tumor cells
  • FAS- or granzyme-, perforin-, and granulysin-mediated killing
TCRγ/δ+ T cells <10% of all T cells in peripheral bloodγ/δ TCR+, CD3, CD4+/–, CD8+/–
  • 'Unconventional' T cell subtype that exhibits antigen-presenting as well as cytotoxic qualities in response to stress antigens
  • Recognition of antigens without the presence of MHC molecules
Natural killer T cells (NKT cells)<3% of all T cells in peripheral bloodCD45, CD3, CD56, CD16, NK1.1, granzyme
  • Heterogeneous group of 'unconventional' T cells that share properties of both T cells and natural killer cells 
  • Recognition of non-polymorphic antigen-presenting CD1d molecule rather than MHC (important in recognizing, e.g., glycolipids of Mycobacterium tuberculosis)
Double-negative T cells1–3% of all peripheral T cellsα/β TCR+, CD3
  • Express α/β TCR, but lack expression of CD4 or CD8, as well as typical NKT cell markers
  • Potential role in down-regulation of the immune response

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.
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Overview of human T cell differentiation from naive to memory T cells (PMID: 24258910, 26999211).

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.

2.2 Miltenyi Biotec application protocols for pan T cells from peripheral blood

Miltenyi Biotec has created dedicated application protocols for the isolation, activation, and expansion of pan T cells.

2.3 Sample preparation of peripheral blood

Miltenyi Biotec offers various kits for the direct isolation of pan T cells from peripheral blood and blood products. No specific sample preparation is needed when using those kits. T cells can also be isolated from peripheral blood mononuclear cells (PBMCs), which can be generated either by density gradient centrifugation or using the MACSprep™ PBMC Isolation Kit, human. For details, see chapter Human peripheral blood.
MACS Handbook:

Blood (human)

2.4 Magnetic cell separation of pan T cells from peripheral blood

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.
2.4.1 Isolation of T cells directly from whole blood, buffy coat, and LRSC

At a glance: Kits and reagents for the separation of pan T cells from whole blood, buffy coat, and LRSC

Starting materialIsolation strategyCommentsAutomationProduct
Whole bloodDepletion of non-target cellsIsolation of pan T cells directly from up to 30 mL whole blood in less than 30 minutes.NoMACSxpress® Whole Blood Pan T Cell Isolation Kit, human
Buffy coatDepletion of non-target cellsIsolation of pan T cells directly from an entire buffy coat in less than 30 minutes.NoMACSxpress® Buffy Coat Pan T Cell Isolation Kit, human
LRSCDepletion of non-target cellsIsolation of pan T cells directly from LRSC in less than 30 minutes.NoMACSxpress® LRSC Pan T Cell Isolation Kit, human
Whole blood Positive selection of target cellsIsolation 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.

Before separation
After separation

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.

The MACSxpress® Whole Blood Pan T Cell Isolation Kit enables isolation of untouched pan T cells directly from larger volumes of whole blood (total capacity 3x30 mL) without density gradient centrifugation. Non-target cells are removed by immunomagnetic depletion using MACSxpress Beads, and erythrocytes are simultaneously sedimented to yield untouched target cells of high purity. Similar kits based on MACSxpress Technology are available for the isolation of Pan T cells from buffy coat or LRSC.
Unseparated fraction
After separation

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.

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2.4.2 Isolation of pan T cells from PBMCs

At a glance: Kits and reagents for the separation of Pan T cells from PBMCs

Starting materialIsolation strategyCommentsAutomationProduct
PBMCsPositive selection of target cellsCD2 is one of the earliest T cell markers, involved in thymic development.Yes*CD2 MicroBeads, human
PBMCsPositive selection of target cellsCD3 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
PBMCsPositive 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.

NoREAlease CD3 MicroBead Kit, human
PBMCsPositive selection of target cellsCD6 is expressed on the majority of T cells, as well as on a subset of B cells.Yes*CD6 MicroBeads, human
PBMCsDepletion of non-target cellsIsolation 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.
PBMCs before separation
Untouched T 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.

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2.4.3 Isolation of naive/memory T cell subsets from PBMCs

At a glance: Kits and reagents for the separation of naive/memory T cell subsets from PBMCs

Starting materialIsolation strategyCommentsAutomationProduct
PBMCsDepletion of non-target cellsDepletion 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
PBMCsPositive selection of target cellsIsolation of naive T cells, both CD4+ and CD8+. CD62L is expressed on all lymphocytes, not only T cells.Yes*CD62L MicroBeads, human
PBMCsPositive selection of target cellsIsolation 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
PBMCsPositive selection of target cellsIsolation 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
The Naive Pan T Cell Isolation Kit, human, provides fast and efficient isolation of untouched naive T cells from human PBMCs. To deplete memory T cells and non-T cells, PBMCs are incubated with a cocktail of biotinylated CD45RO, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD57, CD123, Anti-HLA-DR, CD235a (Glycophorin A), and CD244 antibodies. Optionally, biotinylated Anti-TCRγ/δ antibodies can be added for depletion of TCRγ/δ+ T cells. Subsequently, the cells are magnetically labeled with Anti-Biotin MicroBeads. CD61 MicroBeads are added for concurrent depletion of platelets.
PBMCs before separation
Untouched naive T cells

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.

2.4.4 Isolation of activated T cells

At a glance: Kits and reagents for the separation of activated T cells from PBMCs

Starting materialIsolation strategyCommentsAutomation Product
 PBMCsPositive selection of target cellsCD137 is involved in the activation and survival of CD4+ and CD8+ T cells, and NK cells.Yes*CD137 MicroBead Kit, human
PBMCsPositive selection of target cellsCD154 is up-regulated on activated CD4+ T cells.Yes*CD154 MicroBead Kit, human
PBMCsPositive selection of target cellsCD25, 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
PBMCsPositive selection of target cellsCD30 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
PBMCsPositive selection of target cellsCD69 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. 

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Reliable characterization of functional subsets of the entire BK virus–reactive CD4+ T cell pool. Characterized cells included TNF-α−, IFN-γ−, and IL-2–producing T cells. Cells were first enriched using the CD154 MicroBead Kit, human, according to the rapid ARTE protocol.

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.

2.4.5 Isolation of 'unconventional' T cell subsets

At a glance: Kits and reagents for the separation of activated T cells from PBMCs

Starting materialIsolation strategyCommentsAutomation Product
 PBMCsPositive selection of target cellsDirect immunomagnetic separation via the γ/δ variant of the T cell receptor (TCRγ/δ).Yes*Anti-TCRγ/δ MicroBead Kit, human
PBMCsDepletion of non-target cellsNon-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
PBMCsPositive selection of target cellsPositive 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
PBMCsDepletion of non-target cellsNK 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
PBMCsDepletion of non-target cells followed by positive selection of target cellsCD4CD8CD56CD3+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). However, also other CD1d-restricted T cells ('type 2' NKT) 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. Natural killer T cells may share other features with NK cells, such as CD16 and CD56 expression and granzyme production.

The best-known NKT cell subtype are the invariant natural killer T (iNKT) cells. In humans, these 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+CD4CD8 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 (CD4CD8CD56CD3+TCRα/β+ cells) from PBMCs by sequential sorting. The isolation is performed in a two-step procedure.

PBMCs before separation
Pre-enriched double-negative T cells after depletion of CD4+, CD8+, and CD56+ cells
Isolated double-negative T cells

Isolation of double-negative T cells. TCRα/β+CD4CD8 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.

2.5 Characterization of T cells by flow cytometry

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. 

2.5.1 Flow cytometry panels

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 cellsExhausted T cellsTCRγ/δ+ T cellsNKT cells 
CD3CD3CD3TCR γ/δ CD3
CD4CD4CD4CD3 CD4+/–
CD8CD8CD8CD4+/– CD8+/–
CD27CD25 (IL2RA)CD96 (TACTILE)CD8+/– CD16
CD28CD69CD152 (CTLA-4) CD56
CD45RACD95 (FasR)CD223 (LAG-3)
 CD57
CD45ROCD134 (OX40)CD244 (2B4)
 CD161 (NK1.1)
CD57CD137 (4-1BB)CD272 (BTLA)
 TCR Vα24-Jα18
with TCR Vβ11 (iNKT)
CD62L (L-Selectin)CD154 (CD40L)CD278 (ICOS)
 
CD69Ki-67CD279 (PD1)
 
CD95 (FasR)KLRG1CD366 (TIM-3)
 
CD127TIGIT
 
CD197 (CCR7)VISTA
 
EOMES
 
2.5.2 Analysis of surface markers and cytokines

Miltenyi Biotec offers a range of solutions for the analysis of T cell–associated surface markers and cytokines: 

  • MACS Antibodies are the ideal solution for the staining of T cell surface markers or the intracellular staining of cytokines.
  • MACS Cytokine Secretion Assays allow detection and enrichment of viable cytokine-secreting cells. The human kits are available for a large number of cytokines, including TNF-α, GM-CSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, and IL-22, and can be used for further characterization of T cell subsets.
  • The MACSPlex Cytokine 12 Kit, human is used for multiplex analysis of secreted cytokines in serum and cell culture supernatants using a standard flow cytometer. Cytokines that can be analyzed in a single sample include: GM-CSF, IFN-α, IFN-γ, lL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and TNF-α.
  • The Rapid Cytokine Inspectors allow for rapid analysis of activated T cells by combining surface marker analysis (e.g., CD4, CD8, CD154) with intracellular cytokine staining (e.g. IFN-γ, TNF-α, IL-2, and IL-17).
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Principle of the Cytokine Secretion Assay – Cell Enrichment and Detection Kits.

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Principle of MACSPlex Assays. Samples containing unknown levels of analytes are incubated with the antibody-coated MACSPlex (MPx) Capture Beads, and analytes bind to the specific antibody. The MPx Detection Reagent, composed of a cocktail of APC-conjugated antibodies specific for the analytes, is added. Consequently, sandwich complexes are formed between MPx Capture Bead,  analyte, and MPx Detection Reagent. These complexes can be analyzed based on the fluorescence characteristics of both MPx Capture Bead and Detection Reagent. Standards of known quantities of given analytes are provided with the kit and are used for the quantification of the analytes within the unknown samples.

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2.6 Cell culture of pan T cells

Miltenyi Biotec offers a specialized and versatile range of culture media and reagents for the stimulation, activation/expansion, and differentiation of T cells.

2.6.1 Cultivation, activation, and expansion of T cells

At a glance: Kits and reagents for the cultivation, activation, and expansion of T helper cells

Use Comments Product
Culture mediumOptimized T cell media without serum or animal-derived components. Also available in MACS GMP grade and with or without phenol red.TexMACS Medium
SupplementConsistent, high-quality recombinant cytokines for successful cell culture. Available in premium, research and MACS GMP grades.MACS Cytokines
StimulationNanomatrix-based activation of T cells via CD3/CD28 engagement.Available in research, and MACS GMP grades.T Cell TransAct
StimulationCell-sized activation beads (‘artificial antigen-presenting cells’) loaded with activating CD2, CD3, and CD28 antibodies.T Cell Activation/Expansion Kit, human
StimulationNon-toxic alternative to Staphylococcal enterotoxin B (SEB). Functions as a superantigen.CytoStim
StimulationExtensive 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
StimulationIn vitro T cell activation and expansion.

CD3 pure – functional grade, human

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

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Comparison of expansion rates of human T cells in different media. Cells were expanded by the T Cell Activation/Expansion Kit, human, in combination with TexMACS Medium, a product from another manufacturer, or serum-containing basal medium (RPMI + 10% FBS).

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.

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Activation of human purified T cells. T cells isolated using the Pan T Cell Isolation Kit were activated for 48 hours using T Cell TransAct in TexMACS Medium supplemented with Human IL-2. Cells were fluorescently stained and analyzed by flow cytometry using the MACSQuant® Analyzer.

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.

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Decision tree: T Cell Activation and Expansion Kit vs. T Cell TransAct.

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.

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A comparison of different T cell stimulation reagents. Freshly isolated human PBMCs were stimulated for 4 h using either CytoStim, SEB or PMA/Ionomycin. Cells were subsequently stained with CD4, CD154-APC and Anti-IFN-γ-PE antibodies and analyzed for CD154 and IFN-γ expression after pre-gating for CD4.

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.
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3 T cells from other tissues

T cells can also be obtained from tissues other than peripheral blood. Miltenyi Biotec offers a variety of solutions for the preparation of human tissues, as well as the subsequent separation, cultivation, and analysis of the respective T cell populations.

3.1 Miltenyi Biotec application protocols for T cells from other tissues

3.2 Sample preparation of different 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.