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Manual Magnetic Cell Isolation with Streptamers®

Streptamers®: fully reversible reagents for the selection of uncompromised, authentic cells

Cell biology is reliant on the investigation of unmanipulated and uncompromised cells. IBA's Streptamer® technology is the only fully reversible method for magnetic cell isolation as well as fluorescent cell staining. After removal of the reagents by adding biotin (vitamin H), authenic cells are ready for further experiments!

Choose your field of interest

Streptamer® - Key features

  • no stimulation of cells after cell separation
  • optimal preservation of cell effector function
  • improved viability of cells
  • high reproducibility

Streptamer® - Principle

Example of T cell isolation with magnetic Fab Streptamers®:

Magnetic Fab Streptamers® Magnetic MHC I Streptamers®
for (serial) positive selection(s) of T cells from PBMCs with magnetic microbeads and a hand-held magnet for selection of antigen-specific CD8+ T cells from PBMCs with magnetic nanobeads and magnetic columns
Full reversibility in detail
Low affinity Strep-tagged Fab fragments or MHC I molecules are multimerized with Strep-Tactin®, a streptavidin derivative, to generate multimers with high binding avidity. Strep-Tactin® fluorochromes are used for cell staining / FACS isolation and Strep-Tactin® magnetic beads are used for magnetic cell isolation. Upon addition of low concentrations of biotin (Vitamin H), which competes with high affinity for the binding of Strep-tag to Strep-Tactin®, staining and isolation reagents dissociate rapidly from the cell surface.
This reversibility enables multiple cell stainings with different Strep-Tactin® fluorochromes as well as sequential positive cell selections with magnetic beads.
A modular system for cell selection AND staining

Fab-Streps or MHC-Streps can be combined either with fluorescence- or bead-labeled Strep-Tactin® for cell staining or magnetic isolation, respectively. Biotin is offered as stock solution with the Streptamer® Solution Set which is used for removal of cell staining as well as cell isolation reagents. This modular system enables the combination of multiple receptor- or antigen-specific staining and isolation products, making your experiments more convenient and economic. All reagents can be purchased as single products or assembled in ready-to-use staining or isolation kits.

Streptamer® - Application data

Full reversibility and preserved function of isolated cells

Reversible Streptamers® confer full protection against L. monocytogenes infection in mice (Knabel et al., 2002)

The reversible Streptamers® maintain the in vivo function of isolated cells. In contrast, conventional MHC multimers/tetramers signifcantly change the phenotype and function of stained T cells at physiological temperatures (Knabel et al., 2002).

Pretreatment of LLO91-99-specific T cells with conventional binding MHC tetramer reagents resulted in significantly reduced protection towards Listeria infection following adoptive transfer. In contrast, after complete removal of the Streptamers® with biotin, the same number of cells conferred almost an identical degree of protection as compared with positive controls.

Without removal of the Streptamers® before adoptive cell transfer, the cells were reproducibly more effective than cells coated with conventional MHC tetramers. Please read the complete paper Knabel et al., 2002.

Reversible Streptamers® preserve proliferation capacity and functional status of CD8+ T cells (Wang et al., 2013)

Proliferation of CMVpp65-specific CD8+ T lymphocytes was preserved after selection with reversible Streptamers®, while it might have been altered with tetramers (Fig.3). Furthermore, the functional status as measured by activity (secretion of IFN-gamma) and cytotoxic effectiveness (secretion of granzyme B) remained very active when selected with Streptamers® as compared to tetramers (Fig.4).

Reversible Streptamers® preserve T cell receptor expression of CD8+ T cells (Zhang et al., 2016)

Fab Streptamers® for T cell isolation

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 isolation 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.