Immune cell isolation directly from blood with reversible cell labeling using StraightFrom® REAlease® MicroBeads

REAlease Technology relies on engineered antibody fragments that when in a monomeric state have low affinity for an epitope. 

When multimerized in a biotinylated REAlease Complex, this complex binds to cell epitopes with high avidity. Together with an anti-Biotin MicroBead, the target cell is magnetically labeled. During magnetic isolation, labeled target cells are retained within the column and separated from non-labeled cells.

To get MicroBead-free target cells, the REAlease Bead Release Reagent is applied to the column during the elution step. The eluted target cells are free from anti-Biotin MicroBeads. 

Label-free target cells can be obtained by using the REAlease Release Reagent. This leads to monomerization of the antibody fragments and dissociation of the complex from the cell surface. The result is that cells are free of any label. As their epitopes are available for re-labeling, the cells are suitable for any downstream application.
 

Label free cells with REAlease Biotin Complex

CD3⁺ cells were isolated from a complete buffy coat using the StraightFrom® Buffy Coat REAlease® CD3 MicroBead Kit, Whole Blood Columns, and a MultiMACS Cell24 Separator Plus with the Single-Column Adapter. 

Cells were fluorescently stained with REAfinity Antibodies CD3-PE and Biotin-APC-Vio 770 (for recognition of REAlease Biotin Complex) and analyzed by flow cytometry using the MACSQuant Analyzer X. 

Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. 

Directly after isolation, the cells showed staining of biotin (“MicroBead-free CD3⁺ cells”), whereas the label-free CD3⁺ cells after the REAlease Biotin Complex release were negative for biotin similar to the non-labeled cells before separation.

Level of cell activation

The label-free CD3⁺ T cells obtained with StraightFrom Buffy Coat REAlease CD3 MicroBead kit, human show same level of non-activation (near zero CD25/CD69 expression level) during 2 to 3 days after isolation as the untouched T cells obtained with Pan T cell Isolation kit, human from the same blood material. Furthermore, the label-free and untouched T cells had same level of activation 2 to 3 days after isolation and stimulation (highly increased CD25/CD69 expression level and reduced MFI CD3).

With the same approach, CD4⁺ and CD8⁺ unlabeled cells have shown comparable level of non-activation as untouched cells when unstimulated for 2 days, and also same level of activation 2 days after isolation and stimulation, in comparison of StraightFrom Buffy Coat REAlease CD4 or CD8 Microbead kits, human and CD4⁺ or CD8⁺ T cell isolation kit, human respectively on same buffy coats (results not shown).