Cell labeling for immune cell isolation | Miltenyi

The innovative StraightFrom REAlease Technology skips the time-consuming step of density gradient centrifugation and allows full flexibility with cell labeling. The purified target cells are bead-free, practical for consecutive magnetic isolations, and can be label-free so that epitopes are available for any downstream application. The isolated cells are suitable for flow cytometric analyses, molecular biology applications, and functional studies.

How it works

Basic principle

REAlease MicroBead Technology - The basic principle

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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.

Protocol overview

In the diagram below, we show you the steps and approximate amount of time needed for conventional, StraightFrom MicroBeads, and StraightFrom REAlease MicroBeads protocols.

Conventional protocols require the isolation of PBMCs by density gradient centrifugation before target cell isolation. The innovative StraightFrom REAlease Technology omits this time-consuming step delivering bead-free target cells directly from blood materials within 40 minutes.

The protocol for StraightFrom REAlease MicroBeads is based on positive isolation and the use of REAlease Biotin Complex.

StraightFrom and StraightFrom REAlease MicroBeads protocols simplify and shorten the isolation process to obtain target cells.

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Automated cell separation

Leukocyte subsets can be purified either automatically, using the MultiMACS™ Cell24 Separator Plus or manually using a MACS® Separator.

Automated cell isolation with the MultiMACS™ Cell24 Separator Plus

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The MultiMACS Cell24 Separator Plus is designed to process an entire buffy coat in a single run with StraightFrom® Buffy Coat REAlease® MicroBead kit. The 40-minute isolation procedure results in bead-free cell populations with high purity and recovery rates. With 12 Whole Blood Columns (included in the kit), the instrument allows time saving cell isolation when processing large sample volumes, while providing reliability and reproducibility in day-to-day cell separations.

For fully automated cell separations, the MultiMACS X is the instrument to choose.

Data

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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.

Applications

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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).

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Brochure

StraightFrom MicroBeads - Cell isolation directly from blood products

Download the brochure to get all the details on our StraightFrom MicroBead Technology.

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