Stimulation, magnetic enrichment, and flow analysis of antigen-specific T cells

  • Potent stimulation with PepTivator® Peptide Pools
  • Efficient enrichment of antigen-activated T cells
  • Reliable multiparameter flow analysis of antigen-specific T cells

Application protocols

Discover our different workflows for antigen-specific T cells and find the one that fits your experimental needs.

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Working with antigen-specific T cells - Solutions for immuno-oncology and more

Covered in this webinar are Miltenyi Biotec’s unique solutions for the stimulation, pre-enrichment, isolation, and analysis of these rare cells.

Application data by workflow step

PBMC isolation

Comparison of PBMC populations isolated with the MACSprep PBMC Isolation Kit or by density gradient centrifugation
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Fast PBMC isolation with the MACSprep™ PBMC Isolation Kit, human

  • Untouched isolation of PBMCs from 1–10 mL of human whole blood

  • No density centrifugation, no erythrocyte lysis

  • Fast processing in only 25 minutes

  • Superior reproducibility, recovery, and cell viability

T cell stimulation

Production of IFN-γ by human CD4+ or CD8+ T cells after stimulation with different antigens using PepTivator Peptide Pools
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PepTivator® Peptide Pools

  • Stimulation of both CD4+ and CD8+ T cells with our extensive panel of antigens

  • 15-mer peptides with 11-amino acid overlap that cover the complete sequence of the respective antigen

  • Available in 96-well format

  • MACS GMP-grade peptide pools for translational settings

Enrichment of antigen-specific T cells

Enrichment of antigen-specific CD4+ T cells using the CD154 MicroBead Kit, human
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Isolation of antigen-specific CD4+ T cells with the CD154 MicroBead 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 co-stimulatory molecule in T cell/antigen-presenting cell (APC) interactions through ligation of CD40. Due to its transient expression within hours after activation, CD154 can be used as a marker for isolation of activated antigen-specific CD4+ T cells. Optionally, pre-enrichment can be performed using the CD4+ T Cell Isolation Kit, human.

Isolation of antigen-specific CD8+ T cells with the CD137 MicroBead Kit, human

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 to be 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.  For the isolation of activated antigen-specific CD8+ T cells, the CD137 MicroBead Kit can be combined with the CD8+ T Cell Isolation Kit.

Principle of the MACS Cytokine Secretion Assays
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Cytokine Secretion Assay – Cell Enrichment and Detection Kits

  • Unique technology for the isolation of viable cytokine-secreting cells

  • Accurate analysis of cytokine secretion at single-cell level

  • Sensitive detection of one T cell per million cells

  • Available for human and mouse cells secreting TNF-a, GMCSF, IFN-a, IFN-g, or IL-2, -4, -5, -10, -12, -13, -17, -22

Rapid ARTE – Enrichment of rare antigen-specific CD154+CD4+ T cells for sensitive flow analysis

By combining magnetic cell enrichment and multiparameter flow cytometry analysis of CD154+CD4+ T cells, Rapid ARTE allows the processing of larger cell numbers and the direct ex vivo detection and characterization of rare antigen-specific T cells.

  • Direct ex vivo detection and characterization of rare antigen-specific T cells

  • Fast protocol with only 1 hour hands-on time

  • Unique combination of cell enrichment via CD154 marker and multiparameter flow analysis

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Scientific poster
The rapid antigen-reactive T cell enrichment procedure enables direct ex vivo BKV-specific CD4+ T cell characterization

Michaela Niemöller, Mario Assenmacher, and Anne Richter
Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany

Enrichment of CMV-specific CD8+ T cells using the MACSQuant Tyto Cell Sorter

With a small footprint, gentle sorting, and disposable cartridges, the MACSQuant Tyto Sorter is ideally suited for the isolation of even the rarest ag-specific T cells in both research and clinical settings.

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Application note
Isolation of CD8+ antigen-specific T cells

Find out more about the MACSQuant Tyto’s ability to isolate rare antigen-specific cytotoxic T cells to a purity above 90%.

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Scientific poster

GMP-compliant flow cytometric cell sorting of antigen-specific T cells using MACS® GMP CD8-APC and MACS GMP CD137-PE Fluorescent Antibodies on the MACSQuant® Tyto® Cell Sorter

Antigen-specific T cell isolation for adoptive T cell therapy

The CliniMACS® Cytokine Capture System (IFN-gamma) allows the direct isolation of antigen-specific CD4+ and CD8+ T cells following a short in vitro stimulation period with a specific antigen. 

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Automatic isolation of antigen-specific T cells for adoptive T cell therapy

CD4+ and CD8+ T cells stimulated in vitro with a specific antigen can be magnetically labeled with the CliniMACS Cytokine Capture System (IFN-gamma). In combination with the new CliniMACS Prodigy®, the system allows fast and fully automated generation of antigen-specific T cells in a closed sterile system.

Flow cytometric characterization of antigen-specific T cells

As the final step in many workflows, flow analysis plays an important part in various T cell applications. We provide reagents and detailed application notes that help you make sure your flow analysis and phenotypic characterization of rare antigen-specific T cells are reliable and consistent.

  • REAfinity Recombinant Antibodies recombinantly generated antibodies that ensure lot-to-lot consistency and eliminate background signal
  • Rapid Cytokine Inspector – combined surface marker and intra-cellular cytokine staining for multiparameter analysis of activated human T cells
  • Cytokine Secretion Assay – Detection Kits – flow analysis of cytokine secretion at a detection level of one T cell in a million
  • Build your own T cell flow panel with our Flow Panel Builder
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Application note
Detection, enrichment, and direct phenotypic and functional characterization of antigen-specific naive and memory T cell subsets

Petra Bacher and Alexander Scheffold
Department of Cellular Immunology, Clinic for Rheumatology and Clinical Immunology, Charité, University Medicine Berlin, Berlin, Germany

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