How Does DSP Fixation Preserve Single Cells and RNA?

Researchers studying gene expression and protein interactions at the single-cell level need methods that preserve cellular integrity while maintaining RNA and protein stability. DSP (dithiobis(succinimidyl propionate)) is a crosslinking reagent that stabilizes biomolecules without excessive damage or loss of information.

This method is particularly valuable when spatial context and molecular content must be retained. Understanding DSP fixation helps optimize experimental outcomes and ensures reliable data collection.

Mechanism Of Crosslinking

DSP fixation establishes covalent bonds between biomolecules, stabilizing cellular components while preserving their interactions. As a homobifunctional crosslinker, DSP contains two reactive N-hydroxysuccinimide (NHS) ester groups that target primary amines, mainly in lysine residues of proteins. In aqueous environments, these NHS esters undergo nucleophilic attack, forming stable amide linkages that reinforce protein structures and prevent post-fixation degradation.

A key feature of DSP is its disulfide bond, which allows for reversible crosslinking. Unlike irreversible fixatives such as formaldehyde, DSP’s disulfide linkage can be cleaved under reducing conditions, enabling selective recovery of biomolecules. This property is especially useful in single-cell studies, where controlled reversal of fixation allows molecular analyses while maintaining structural fidelity.

The efficiency of DSP crosslinking depends on concentration, reaction time, and buffer composition. Optimal fixation occurs within a 0.5–2 mM concentration range, with incubation times of 10–30 minutes at physiological pH. Buffers containing primary amines, such as Tris, interfere with NHS ester reactivity, making phosphate-buffered saline (PBS) a better choice. Temperature also affects reaction kinetics, as higher temperatures accelerate NHS ester hydrolysis and can reduce fixation effectiveness if not carefully managed.

Preserving RNA And Proteins

Preserving RNA and proteins during DSP fixation is essential for accurate molecular analyses. RNA is highly susceptible to degradation by ribonucleases (RNases), but DSP rapidly crosslinks proteins, including RNases, reducing their activity and protecting RNA. This stabilization is particularly valuable in single-cell applications, where even minor RNA degradation can compromise gene expression profiling. Studies show that DSP fixation retains RNA integrity comparable to unfixed controls when processed under optimal conditions.

DSP also preserves protein structure and antigenicity, which is crucial for proteomic studies. By forming covalent bonds between lysine residues, DSP prevents protein unfolding and degradation while maintaining intracellular organization. Unlike harsh fixatives that induce protein denaturation, DSP maintains epitope accessibility, making it compatible with immunostaining and mass spectrometry-based proteomics. This is particularly useful in spatial transcriptomics and multiplexed protein analyses, where preserving native molecular interactions is necessary for accurate data interpretation.

DSP’s reversibility further enhances biomolecule preservation by enabling selective recovery of RNA and proteins. Under reducing conditions, the disulfide bonds within DSP are cleaved, releasing crosslinked molecules. This allows researchers to extract high-quality RNA for sequencing while retaining protein interactions. Comparative studies show that RNA extracted from DSP-fixed cells has high yield and integrity, with minimal fragmentation, making it suitable for single-cell RNA sequencing (scRNA-seq) and other transcriptomic techniques.

Effects On Cell Morphology

DSP fixation maintains cell morphology by reinforcing structural integrity without causing excessive shrinkage or distortion. Unlike harsh fixatives that alter cellular architecture, DSP preserves intracellular organization through covalent crosslinking. This prevents cytoskeletal collapse, which is important for analyzing spatial relationships between organelles. High-resolution microscopy studies show that DSP-fixed cells retain their shape and compartmentalization, making them suitable for imaging-based analyses.

DSP also helps preserve membrane integrity, which can be compromised by fixatives that extract or aggregate lipids. By crosslinking membrane-associated proteins, DSP maintains plasma membrane continuity and prevents artifacts that obscure subcellular structures. This is especially beneficial for electron microscopy, where maintaining ultrastructural detail is critical. Researchers have observed that DSP-fixed cells exhibit well-preserved nuclear and cytoplasmic boundaries, ensuring that morphological features remain distinguishable at nanometer resolution.

Excessive crosslinking can lead to cellular compaction, affecting downstream applications that rely on accurate size measurements. DSP’s reversible nature mitigates this issue by allowing controlled fixation without excessive rigidity. Comparative analyses show that DSP-fixed cells exhibit minimal volumetric changes compared to unfixed controls, preserving their dimensions for quantitative studies. This is particularly relevant in single-cell imaging and flow cytometry, where maintaining accurate cell size is necessary for proper data interpretation.

Handling Considerations

Proper handling of DSP fixation ensures consistent results while minimizing technical challenges. DSP rapidly degrades in aqueous solutions, so it is typically dissolved in anhydrous dimethyl sulfoxide (DMSO) or other non-aqueous solvents immediately before use. Delayed application or prolonged exposure to moisture leads to premature hydrolysis, reducing crosslinking efficiency. Researchers must account for DSP’s short half-life in solution, requiring careful timing and preparation.

Buffer choice significantly affects DSP’s performance. Tris-based buffers contain primary amines that react with DSP’s functional groups, reducing crosslinking efficiency. To avoid this, phosphate-buffered saline (PBS) or HEPES buffers are recommended, as they do not interfere with NHS ester chemistry. Maintaining a physiologically relevant pH (7.2–7.6) helps preserve cellular conditions during fixation and minimizes unintended artifacts.

Temperature also influences DSP’s reaction kinetics. Elevated temperatures accelerate NHS ester hydrolysis, shortening the effective fixation window, while excessively low temperatures slow reaction rates, potentially leading to incomplete crosslinking. Most protocols recommend performing DSP fixation at room temperature or on ice, depending on the application. When working with single-cell suspensions, gentle mixing ensures uniform fixation without introducing mechanical stress that could compromise cell integrity.

Suitability For Single-Cell Studies

DSP fixation is well-suited for single-cell studies due to its ability to preserve molecular integrity while allowing controlled reversibility. It is particularly valuable in single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and protein-protein interaction studies. Researchers working with heterogeneous cell populations benefit from DSP’s ability to stabilize cellular components without introducing artifacts that could affect downstream analyses. This is especially relevant for studying rare cell types, where sample preservation is critical.

A major advantage of DSP fixation in single-cell applications is its compatibility with multi-omic approaches. Techniques that require both transcriptomic and proteomic analyses from the same sample rely on fixation methods that do not interfere with molecular extraction. DSP’s reversible crosslinking allows for selective RNA recovery while maintaining protein interactions, enabling researchers to study gene expression alongside protein localization. Studies show that DSP-fixed single cells retain high RNA integrity, with minimal loss of transcript diversity compared to fresh samples. This is particularly beneficial in clinical settings, where preserving patient-derived single-cell samples for extended periods is necessary before processing.