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Cell Isolation

Cells serve as an important research tool to investigate different mechanisms in health and disease. They are also suitable for diagnostic and therapeutic purposes, making them attractive for a broad area of research fields. Immune cells have a wide range of functions such as controlling body homeostasis, which includes elimination of infected or cancerous tissue. Therefore, these cells are frequently employed for different analyses and model systems. The advantage of using immune cells is that they are abundant in, for example, human blood or mouse spleen, two sources that are relatively easily accessible for research purposes.

The interaction of cells with each other is very complex and for some experimental setups it is necessary to isolate one specific population for further downstream applications and analyses. Those include for example DNA/RNA isolation, single cell RNA sequencing, protein purification, Western blot or various cell culture experiments.

Immune cells commonly found in human blood

Immune cells commonly found in human blood.

Positive and negative cell selection

Specific cells are most frequently isolated by targeting their surface markers or according to their antigen-specificity. For surface marker-specific cell isolation, generally one of these two approaches is used: positive or negative cell selection. Antigen-specific cell isolation usually requires a positive cell selection approach.

Principle of positive cell isolation
Principle of negative cell isolation

Cell isolation is possible via positive or negative cell selection. In positive cell selection, cells are directly labeled with e.g., magnetic bead-conjugated antibodies. In negative selection, all unwanted cells are labeled, leaving target cells completely “untouched”.

Surface marker-specific cell isolation

In positive selection, the target cells are directly labelled with, for example, antibodies conjugated to magnetic beads or fluorophores. The cells can subsequently be isolated using a magnet (magnetic-activated cell sorting - MACS) or a flow cytometer suitable for fluorescence-activated cell sorting (FACS).

If the protein that targets a surface marker of a cell, which can be an antibody, a Fab fragment or another cell-binding protein, is fused to a Strep-tag®II or Twin-Strep-tag®, it can be combined with different Strep-Tactin® conjugates to facilitate positive cell selection.

It is possible to choose between affinity chromatographic, magnetic and fluorescent isolation methods depending on the Strep-Tactin® backbone (Strep-Tactin® TACS Agarose columnsStrep-Tactin® Magnetic Microbeads or Strep-Tactin® PE/APC) and is therefore adaptable to different experimental requirements. 

The interaction between Strep-Tactin® and the Twin-Strep-tag® is reversed by biotin addition. This way, Strep-Tactin® reagents can easily be removed from the cell surface and do not affect further downstream procedures.

Different methods for positive cell selection

Cell isolation using positive selection is possible via three principal approaches: fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS) or affinity chromatography. In all approaches, different molecules that target a cell can be used such as antibodies or Fab fragments.

In negative selection, the unwanted cells are labelled usually with antibodies conjugated to magnetic beads. The labelled cells are subsequently separated from the target cells using a magnet. To effectively use the negative selection approach, the composition of a sample has to be known to target all unwanted cells. Therefore, a standard problem of this method is limited purity and recovery, which causes variable quality. The advantage of this method is that cells remain completely “untouched” during the isolation procedure. However, highly pure populations can best be obtained by using positive separation techniques. 

In this application note we demonstrate how Strep-Tactin® TACS Agarose columns can be used for negative cell selection.

Antigen-specific cell isolation

Using the Strep-tag® technology, cells can also be isolated according to their antigen specificity. This becomes especially relevant for characterizing B or T cell mediated adaptive immune responses. Instead of high affinity antibodies, antigens (B cells) or major histocompatibility complexes (MHCs) that present a specific peptide/antigen (T cells) are utilized to select cells of interest.

For antigen-specific T cell isolation, MHC complexes need to be multimerized on a backbone to efficiently capture cells. Combining MHC I molecules fused to a Twin-Strep tag® with Strep-Tactin® backbones conjugated to magnetic microbeads or fluorophores also allows magnetic-activated as well as fluorescence-activated cell sorting in this system.

Similar as for T cells, strep-tagged antigens can be bound to Strep-Tactin® conjugates to enable antigen-specific B cell selection.

The reversible labelling principle that is applied in all our isolation approaches helps to preserve the authentic properties, full effector function as well as viability of the target cells.