FAQ Blog Series: What’s the Difference between Positive and Negative Immunomagnetic Cell Separation?
The simplicity and speed of immunomagnetic cell separation has made it the go-to method for isolating target cell populations since it was pioneered in 1990 by Miltenyi Biotec. The ability to isolate specific cell types from heterogeneous tissue or blood-derived sources such as whole peripheral blood, bone marrow, or cord blood is critical to advancing fundamental research, disease diagnostics, drug discovery and therapeutic development.
For example, human peripheral blood mononuclear cells (PBMCs) can be collected via leukapheresis, in the form of leukopaks, and then further purified to isolate important immune cell subsets, such as T and NK cells, for downstream analyses (i.e., DNA/RNA isolation, single cell sequencing, protein purification) or manipulation (i.e., CRISPR gene editing). In general, cell isolation methods use either a positive or negative cell separation approach, but what’s the difference? And which method should you choose? We’ll look at these questions and more in this installment of the FAQ Blog Series.
Positive vs. Negative Selection: What’s the Difference?
Positive selection: During positive immunomagnetic selection, the target cell population is directly labeled with an antibody linked to a magnetic particle that targets a specific cell surface protein. The cell sample is placed within a magnetic field, which retains the antibody-labeled cells while the fraction containing the unwanted cell types elutes through before the target cells are collected. Here, the target cells remain bound to the conjugated antibodies after selection, which will require additional processing to remove for downstream applications.
Negative selection: Conversely, during negative immunomagnetic cell separation, the unwanted cell populations are labeled for removal with antibody-conjugated magnetic particles targeting specific cell surface proteins—typically a mixture of antibodies are used because there are multiple cell types that need to be removed. The desired cell population remains unlabeled so when the sample is placed within a magnetic field, the target cells will be in the eluted fraction while the unwanted cells are retained.
What Are the Advantages and Disadvantages of Each Method?
Both positive and negative selection approaches have their advantages and disadvantages, which are highlighted below:
|Isolated cells are highly purified
since they are specifically targeted
by antibodies (>90% purity) with a
higher yield potential.Flowthrough or negative fraction
can be put through additional
rounds of cell separation to
isolate other target cell populations.
|Isolated cells remain unlabeled and
are not bound by the conjugated
antibodies leaving the cells unaltered.Protocols are faster and easier to
execute compared to positive
selection with minimal sample
manipulation (closer to in vivo state).
|The positively selected cells remain
bound to the conjugated antibodies,
which may interfere with downstream
assays (i.e., binding assays).
|The cell purity from negative separation
is typically lower because it is more
difficult to target all the unwanted cells
with antibody cocktails compared to a
single target population.
How Do I Choose a Cell Separation Method?
The choice to use a positive or negative selection method is highly dependent on both your target cell population and your research application or goals. Parameters such as purity, yield, and functionality may also influence your decision.
For instance, could having the conjugated antibodies bound to your target cells affect downstream assays or research applications? If yes, then negative selection methods may be preferred. Are you looking for a highly purified cell population? If yes, then positive selection using a targeted antibody is probably best for high purity. Are you interested in isolating more than one cell population? If yes, positive immunomagnetic selection allows for sequential isolation of cells since the flowthrough fraction can be put through secondary rounds of immunomagnetic separation to isolate additional cell types from the same cell source.
AllCells’ Isolated Cell Offerings
It’s clear that there are many influencing factors that determine which approach is best. Alternatively, there are commercially available isolated cell products that can help save valuable time and resources. AllCells offers both positive and negative isolated cell types from leukopaks and bone marrow including T cells (CD3+ and other T cell subsets), B cells, NK cells, and monocytes that come with rigorous quality assurances. AllCells’ employs optimized protocols for each cell type using state-of-the-art equipment and industry best practices resulting in our consistent products that have high recovery and viability.
AllCells has bulk isolation and sequential separation capabilities to suit a variety of research needs—chat with us to find out how we can accelerate your research today!
Learn more about AllCells’ isolated cell products.