Fab-TACS® Gravity - Manual Chromatography Columns for Cell Selection
General workflow using Fab-TACS® Gravity column
The Fab-TACS® Gravity column is filled with a Strep-Tactin® coated matrix made of cell-grade agarose. Strep-tagged Fab fragments (Fab-Streps) specifically bind to the matrix. Subsequently, whole blood or other blood preparations pass through the column. Target cells adhere to the matrix based on the exclusive binding of the Fab-Strep to the target cell. Non-target cells are efficiently washed away. In a final step, the addition of biotin causes the elution of the target cells and the Fab-Streps. After elution, the Fab-Streps self-dissociate from the cell surfaces. The label-free authentic target cells are now ready for further downstream applications.
Most common positive cell selection procedures require the preparation of the PBMC fraction prior to final target cell selection (A). In contrast, the Fab-TACS® technology allows direct cell isolation from whole blood, which saves time (at least 90 min for a PBMC preparation) and minimizes the loss of target cells (B). Here we measured the percentage of target cells using the common selection method or Fab-TACS® Gravity column (C).
After PBMC preparation, nearly 50 % of target cells are lost. Further cells get lost during specific cell selection from PBMC. The Fab-TACS® technology allows cell isolation directly from whole blood, which increases the yields of target cells significantly.
Fab-TACS® Gravity - Application data
The Fab-TACS® technology provides vital benefits for your purified cells and following applications. In this section, we demonstrate the Fab-TACS® benefits with convincing data:
Efficient selection of target cells are important to save resources, money and time!
Fab-TACS® provides high cell yields, because no PBMC preparation is required. Apply your whole blood sample directly to the Fab-TACS® Gravity column!
Figure: CD3+ and CD4+ positive cells were enriched from whole blood samples using the Fab-TACS® Gravity Isolation Kit. The bar chart indicates the yield as mean values of isolated target cells from five independent samples. Fab-TACS® technology enables high yields in a highly reproducible manner.
Fab-TACS® ensures highly pure cells due to highly specific Fab fragments and the underlying positive selection manner.
Figure: Isolation of CD3+ and CD4+ cells directly from whole blood. Cells were stained either with CD3-APC (OKT-3) or CD4-PE (OKT-4) and analyzed by flow cytometry. Dead cells were excluded from analysis using DAPI staining. Doublet and debris discrimination were performed using different FSC/SSC signals. Fab-TACS® technology ensures highest purity independent of the starting material.
Viability is crucial for downstream applications and especially for long-term experiments. The reversible character of Fab-TACS® ensures highly viable cells.
Figure: Viability of isolated cells is shown as mean value of five independent samples. Fab-TACS® technology ensures highly viable and immuno-competent cells for all upcoming downstream applications.
Activation status of Fab-TACS® vs. conventional microbeads selected cells
Cells selected via conventional method with CD3 microbeads or CD3 Fab-TACS® were stained using CD69 (early activation marker) and CD3 antibodies. PBMCs stimulated by PMA/Ionomycin served as positive control. The results indicate that already after 2 hours in culture, the antibody selected cells were activated while cells obtained via Fab-TACS® technology remained unactivated even after 17 hours.
Comparison of cell pellets selected using Fab-TACS® technology or conventional antibody-coated microbeads
The cell pellets remained clear after isolation with Fab-TACS® Gravity column (left), demonstrating its outstanding reversibility. On the contrary, cells isolated via competitor’s conventional microbeads (brown) stayed attached (right), indicating that the selection reagents could not be removed completely.
APC-Fluorescence after Fab-TACS® Gravity CD3 isolation with CD3-FITC and Strep-Tactin®-APC. The target cells do not bind the staining reagent since all isolation reagents dissociated from the cell. Intentionally labeled cells bind the dye resulting in a fluorescence shift. Fab-TACS® ensures authentic target cells.