Preserving the True Immune Landscape in Inflammation Research
Inflammation is a critical process involving complex interactions between innate and adaptive immune cells, stromal populations, and the surrounding tissue microenvironment. Triggered by infection, injury, or dysregulated signaling, inflammation serves to eliminate harmful stimuli and initiate repair. Yet persistent or maladaptive inflammation enables chronic disease, including autoimmunity, cardiovascular disorders, neurodegenerative conditions, and cancer.
Deciphering inflammation at the single-cell level is essential for mapping the cell-cell interactions underlying disease mechanisms that can be targeted for therapeutic interventions. Advances in flow cytometry and single-cell RNA sequencing (scRNA-seq) now allow unprecedented resolution in defining cellular phenotypes, activation states, and signaling networks. However, these technologies depend on a critical assumption: that the cells being analyzed truly reflect their in-situ biology.
Cell Sample Preparation: The Critical Variable
The first step in most single-cell workflows is dissociating tissue into single-cell suspensions. Conventional methods rely on enzymatic digestion (e.g., collagenases, proteases, DNases) to degrade extracellular matrices. While efficient at liberating cells, these methods often compromise biological fidelity.
Evidence of Enzymatic Artifacts
Multiple studies demonstrate that enzymatic digestion alters both surface marker integrity and transcriptional states:
For inflammation research — where identifying subtle immune subset differences (e.g., classical vs. non-classical monocytes) and transient activation states is critical — these artifacts can obscure biological truth and overlook key immune cell interactions.
Acoustic Dissociation: Preserving Biology at the Point of Isolation
The Cellsonics SimpleFlow™ system applies controlled acoustic energy to dissociate tissues without enzymes or elevated temperature. This gentle, acoustic-generated movement preserves epitope integrity, maintains receptor conformation, and minimizes induction of artificial stress responses.
Key Advantages of acoustic dissociation for Inflammation Research
Demonstrating Marker preservations across multiple tissues with acoustic dissociation
Figure 1. Enzymatic dissociation skews macrophage marker detection and phenotype compared to acoustic processing.
Flow cytometry plots show macrophages (MerTK⁺, CD64⁺) from mouse spleen prepared with enzymatic (left) or acoustic (SimpleFlow™, right) dissociation. Enzymatic treatment shifts the macrophage population with altered CD64 and MerTK staining, consistent with cleavage, masking, or induction of marker expression. MerTK, enriched in reparative M2 macrophages, can be upregulated by enzymatic dissociation through release of M-CSF and IL-10, promoting an artifactual M2-like phenotype. Similarly, CD64 (FcγRI), a high-affinity IgG receptor that mediates immune complex clearance and supports inflammation resolution, may be upregulated due to enzyme-induced macrophage activation.
Importantly, the spleen cells were pulled from healthy mice under normal physiological conditions , so minimal macrophage stimulation would be expected.
Figure 2. Acoustic dissociation preserves CD19 marker integrity compared to enzymatic digestion.
Figure 3. Enzyme-free dissociation preserves CD138 expression on B lineage cells.
Median fluorescence intensity (MFI) of CD138, a plasma cell marker, was compared across two dissociation methods in mouse spleen.
These results demonstrate that plasma cell markers like CD138 are particularly vulnerable to enzymatic degradation, which can lead to underestimation of antibody-secreting cell populations in inflamed tissues. By preserving CD138 expression, SimpleFlow™ enables more accurate phenotyping of plasma cells and their contribution to immune responses in inflammation and cancer.
Figure 4. Enzymatic dissociation reduces CD45 detection across immune cells.
Median fluorescence intensity (MFI) of CD45, a pan-leukocyte marker, was compared after acoustic and enzymatic dissociation from murine spleen tissue.
Because CD45 is critical for identifying and gating total leukocytes, enzymatic degradation can bias downstream analyses of immune composition, leading to underestimation of cell recovery in inflamed or tumor tissues. In contrast, SimpleFlow™ acoustic dissociation maintains CD45 integrity, ensuring accurate quantification of the entire immune compartment.
Conclusion
Accurate characterization of the immune landscape in inflammation research depends on preserving cellular phenotypes, interacting cells and activation states at the point of isolation. Traditional enzymatic digestion, while efficient, introduces artifacts that obscure true biology— degrading critical markers, altering phenotypes, and inducing artificial stress signatures. The Cellsonics SimpleFlow™ acoustic dissociation system overcomes these limitations by maintaining epitope integrity, supporting broad immune cell recovery, and minimizing transcriptional artifacts. By enabling researchers to resolve fragile and rare populations, discriminate subtle activation states, and capture authentic immune dynamics, SimpleFlow™ ensures that single-cell analyses reflect in-situ biology. Preserving the true immune landscape not only enhances data quality but also accelerates discovery of new therapeutic targets and strategies across autoimmune, infectious, and cancer immunology.
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