Root > Applications > Cell Selection & Expansion > Manual cell selection > Application notes for Fab and MHC I Streptamers

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Fab Streptamers® for selection of lymphocytes

Sequential positive selection of human CD3+ CD4+ T cells from whole blood with the reversible Fab Streptamers®

PBMCs were isolated with a Ficoll gradient from whole blood. In the first selection step, the CD3 Fab Streptamer® Isolation Kit MB, human  (6-8000-201) was used: PBMCs were incubated with CD3 Fab-Streps conjugated to magnetic microbeads in order to pre-select CD3+ cells. The resulting positive fraction was then treated with D-biotin and washed to remove all labeling reagents. In the second selection step, the CD4 Fab Streptamer® Isolation Kit MB, human  (6-8000-206) was used: CD4+ cells were enriched from the pre-selected CD3+ T cell pool with CD4 Fab-Streps conjugated to magnetic microbeads. Live cells of each selection step are shown. Dead cells were excluded from the analysis with DAPI.

Serial positive selections of lymphocytes and rare subpopulations with the reversible Fab Streptamers

Application Cell Marker, human Application Note
Positive isolation of human
CD3 T cells
CD3 Application Note CD3, pdf
Positive isolation of human
CD8 Cytotoxic T cells
CD8 Application Note CD8, pdf
Tandem purification of human
low frequency antigen-specific
CD8+ T cell subsets
CD8 and MHC I peptides Application Note tandem, pdf
Multiparameter cell selection
of human Central Memory T cells
CD8, CD62L, CD45RA- Application Note Tcm, pdf
Discriminate between human
Naive and Memory (CD45RA-) T cells
CD8, CD62L, CD45RA- Application Note Tcm, pdf
Triple positive isolation of human
Regulatory T cells
CD4, CD25, CD45RA Application Note Treg, pdf
Helper T cell CD4  
MHC I Streptamers® for selection of CD8+ T cells

Staining of antigen-specific CD8 T cells with reversible MHC I Streptamers

High staining intensities with MHC I Streptamers®

High staining intensity with Streptamers® guarantees optimal detection of small T cell populations. Ex vivo staining of CMV-specific CD8+ T cells with HLA-A*0201 CMV pp65495-503 Streptamers® (right diagram) or conventional multimers (left diagram). Staining was performed from a CMV positive individual at 4 °C.

Streptamer® reagents:

  • 6-7001-001  MHC I-Strep CMV, pp65495-503
  • 6-5000-001  Strep-Tactin® PE
  • 6-5603-005  Streptamer® Solution Set Standard for washing and dissociation

Staining and removal of staining from antigen-specific mouse CD8+ T cells

Staining of bacteria-specifc (Listeria monocytogenes, epitope LLO91-99) CD8+ T-cells with H2-Kd/LLO91-99 Streptamers®. Antigen-specific cells were visualized with Strep-Tactin® PE coupled to MHC I-Strep H2-Kd specific for LLO91-99 . After addition of 1 mM d-biotin, subsequent dissociation of monomerized MHC molecules was monitored at different time points as indicated. 20 minutes after biotin addition, staining reagents were largely removed from the cells. Staining and dissociation were performed at 4 °C.

Streptamer® reagents:

  • 6-7014-001  MHC I-Strep H2-Kd, LLO91-99
  • 6-5000-001  Strep-Tactin® PE
  • 6-5603-005  Streptamer® Solution Set Standard for washing and dissociation

 

Staining and removal of staining from antigen-specific human CD8+ T cells

Staining of Her2/neu-specifc CD8+ T cells (kindly provided by H. Bernhard, TU Munich) with HLA-A*0201/Her2/neu369-377 Streptamers as compared to conventional multimers (left diagram). Complete dissociation of Streptamer® staining reagents from the cells is shown after a brief incubation (30 min.) in the presence of 1 mM d-biotin (right diagram). Antigen-specific T cells were visualized with Strep-Tactin® PE coupled to MHC I-Strep HLA-A*0201 specific for Her2/neu369-377. Staining and dissociation were performed at 4 °C.

Streptamer® reagents:

  • 6-7008-001  MHC I-Strep HLA-A*0201, Her2/neu369-377
  • 6-5000-001  Strep-Tactin® PE
  • 6-5603-005  Streptamer® Solution Set Standard for washing and dissociation

All experiments were performed in close cooperation with D. Busch, TU Munich.