Recombinant Mouse Interleukin-2 (Il2) (Active)

What is Recombinant Mouse IL-2 and what structural properties characterize it?

Recombinant Mouse IL-2 is a cytokine originally described for its potent ability to modulate lymphocyte reactivity and promote long-term in vitro culture of antigen-specific effector T lymphocytes . The mature mouse IL-2 protein contains 149 amino acid residues with a molecular weight of approximately 17.2-17.6 kDa . The common commercial form represents the sequence from Ala21 to Gln169, with or without an N-terminal methionine .

Mouse IL-2 exhibits strain-specific heterogeneity in its N-terminal region, which contains a poly-glutamine stretch that can vary between mouse strains . This heterogeneity may have functional implications in experimental settings. The three-dimensional structure consists of a four alpha-helix bundle motif typical of the cytokine family . For optimal bioactivity, proper folding of the protein is essential, with expression systems significantly impacting the final protein structure.

How does mouse IL-2 compare structurally and functionally to IL-2 from other species?

Mouse IL-2 shares 56% amino acid sequence identity with human IL-2 and 73% with rat IL-2 . Despite these sequence differences, mouse and human IL-2 exhibit cross-species activity, indicating conservation of functional domains responsible for receptor binding and signaling . This cross-reactivity makes mouse models valuable for studying certain aspects of human IL-2 biology.

The IL-2 receptor complex is highly conserved and consists of three subunits that are present on cell surfaces in varying preformed complexes . These include:

• The 55 kDa IL-2Rα (CD25), which is specific for IL-2 and binds with low affinity

• The 75 kDa IL-2Rβ (CD122), which is also a component of the IL-15 receptor and binds IL-2 with intermediate affinity

• The 64 kDa common gamma chain (γc/IL-2Rγ), which is shared with receptors for IL-4, IL-7, IL-9, IL-15, and IL-21

Signal transduction is performed by both IL-2Rβ and γc components upon ligand binding . These receptor components are conserved across species, enabling the observed cross-reactivity between mouse and human IL-2.

What expression systems are used for producing Recombinant Mouse IL-2, and how do they affect protein quality?

Two main expression systems are commonly used for recombinant mouse IL-2 production, each with distinct implications for protein quality:

• E. coli expression system: Most commercial recombinant mouse IL-2 is produced in E. coli . This bacterial expression system provides high yields but lacks the capacity for certain post-translational modifications. E. coli-derived mouse IL-2 typically spans amino acids Ala21-Gln169, with or without an N-terminal methionine .

• Pichia pastoris (yeast) expression system: This eukaryotic system enables natural protein folding and post-translational modifications, resulting in greater functionality compared to E. coli-expressed proteins . Yeast-expressed IL-2 more closely resembles the native form of the protein.

The expression system choice directly impacts experimental outcomes. Researchers should select the appropriate system based on their specific experimental requirements, particularly when post-translational modifications may affect protein function or when studying interactions with complex cellular systems.

What are the proper storage and reconstitution methods for Recombinant Mouse IL-2?

Proper storage and reconstitution are critical for maintaining IL-2 bioactivity. The following methods are recommended based on formulation:

For lyophilized preparations (with carrier protein):

• Reconstitute at 100-200 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• Upon initial thawing, aliquot into polypropylene microtubes and store at -80°C for future use

• Avoid repeated freeze-thaw cycles, which can degrade the protein

For lyophilized preparations (carrier-free):

• Reconstitute at 100-200 μg/mL in sterile deionized water

• For long-term storage, add carrier protein (0.5-10 mg/mL) to stabilize the solution

• Store at -80°C in a manual defrost freezer

The protein should not be diluted to less than 10 μg/mL for long-term storage to prevent activity loss . Failure to add carrier protein or store at the indicated temperatures may result in decreased bioactivity.

How is the bioactivity of Recombinant Mouse IL-2 measured, and what are typical activity values?

The bioactivity of recombinant mouse IL-2 is typically measured using cell proliferation assays with the CTLL-2 mouse cytotoxic T cell line, which is IL-2 dependent . The standard measure of potency is the ED50 (effective dose for 50% maximal response), which typically ranges from 0.04-0.4 ng/mL for high-quality recombinant mouse IL-2 .

A typical bioassay protocol involves:

• Serum-starving CTLL-2 cells for 4 hours

• Preparing serial dilutions of the recombinant IL-2

• Incubating 5 × 10³ cells with IL-2 dilutions for 48 hours

• Assessing proliferation using colorimetric assays (e.g., MTT, XTT) or direct cell counting

The ED50 value serves as a critical quality control measure and should be considered when comparing results across experiments or when switching between different sources of recombinant IL-2. Lower ED50 values indicate higher potency, with values consistently below 0.5 ng/mL considered indicative of high-quality preparations.

What are the key differences between carrier-free and BSA-containing Recombinant Mouse IL-2?

The choice between carrier-free and BSA-containing IL-2 can significantly impact experimental outcomes:

BSA-containing IL-2:

• Enhanced protein stability and increased shelf-life

• Allows storage at more dilute concentrations

• Recommended for cell/tissue culture applications and as ELISA standards

• BSA may interfere with certain applications through non-specific binding or by contributing background signal

Carrier-free IL-2:

• Recommended for applications where BSA might interfere with experimental outcomes

• Essential for quantitative mass spectrometry analysis

• Preferred for in vivo studies to avoid potential immune responses to foreign carrier proteins

• Typically less stable and may require more careful handling

When selecting between these formulations, researchers should consider:

• The sensitivity of detection methods to carrier proteins

• The potential for carrier protein effects on the biological system under study

• The requirement for precise quantification of IL-2 concentrations

• The compatibility with downstream applications

What are the optimal concentrations of Recombinant Mouse IL-2 for different in vitro research applications?

The optimal concentration of recombinant mouse IL-2 varies significantly depending on the specific application and cell type:

For T cell proliferation assays:

• 1-10 ng/mL is typically sufficient to support robust proliferation

• The ED50 for CTLL-2 cell proliferation is 0.1-0.4 ng/mL

For regulatory T cell (Treg) expansion:

• Higher concentrations (10-100 ng/mL) may be required

• IL-2 plays a central role in the expansion and maintenance of regulatory T cells

For differentiation of specific T cell subsets:

• Concentrations vary based on the specific subset

• For Th1 differentiation: 5-20 ng/mL

• IL-2 inhibits the development of Th17 polarized cells, so lower concentrations (0.1-1 ng/mL) may be used when Th17 responses are of interest

For use as an ELISA standard:

• Carrier protein concentrations of 5-10 mg/mL are recommended

• For in vitro biological assays, carrier protein concentrations of 1 mg/mL are suggested

Researchers should optimize IL-2 concentrations for their specific experimental systems through dose-response studies, as cellular responses can vary based on activation state, receptor expression levels, and the presence of other cytokines.

How does the Cys160Ser mutation variant of mouse IL-2 differ from the wild-type in research applications?

The Cys160Ser mutation in mouse IL-2 represents a strategic substitution where a cysteine residue at position 160 is replaced with serine . This mutation has several implications for research applications:

• Enhanced stability: The mutation reduces the potential for aberrant disulfide bond formation, resulting in more consistent protein folding and increased stability.

• Bioactivity comparison: The ED50 for the Cys160Ser variant in CTLL-2 proliferation assays is 0.04-0.24 ng/mL , which is comparable to or slightly more potent than wild-type IL-2 (0.1-0.4 ng/mL) .

• Reduced aggregation: The variant typically shows less tendency to form aggregates during storage and use, which can be advantageous for certain applications requiring monomeric protein.

• Experimental consistency: The more stable nature of the Cys160Ser variant may provide greater batch-to-batch consistency in experimental outcomes.

When deciding between wild-type and Cys160Ser variants, researchers should consider whether their experimental question requires native IL-2 structure or whether the enhanced stability of the mutant form would be advantageous. For most in vitro applications, the mutant form provides comparable bioactivity with potential practical advantages.

What are key considerations for troubleshooting inconsistent results when using Recombinant Mouse IL-2 in cell culture?

When encountering inconsistent results with recombinant mouse IL-2 in cell culture, researchers should consider the following factors:

• Protein quality and storage:

  • Check for protein degradation due to improper storage or repeated freeze-thaw cycles

  • Verify bioactivity using a standard CTLL-2 proliferation assay

• Receptor expression on target cells:

  • Confirm expression of all three IL-2 receptor components (IL-2Rα, IL-2Rβ, and γc) on target cells

  • Activation status of cells may influence receptor expression and responsiveness to IL-2

• Interfering factors in culture medium:

  • Serum components may contain IL-2 inhibitors or alternative growth factors

  • Presence of other cytokines may synergize with or antagonize IL-2 effects

• Experimental conditions:

  • Cell density can impact IL-2 consumption rates and apparent activity

  • Timing of IL-2 addition relative to other stimuli is critical for certain responses

  • IL-2 may have different effects depending on the activation state of T cells

• Technical considerations:

  • Verify proper reconstitution procedure was followed

  • Ensure carrier proteins were pre-screened for possible effects in your experimental system

  • Check for high endotoxin levels in preparations that could confound results

Systematic evaluation of these factors can help identify the source of inconsistency and lead to more reproducible outcomes in IL-2-dependent experiments.

How does IL-2 signaling pathway function in T cells and what downstream effects can researchers expect to observe?

IL-2 signaling in T cells follows a complex pathway with multiple downstream effects that researchers should consider in experimental design:

The IL-2 receptor complex consists of three subunits with varying affinities and expression patterns :

• IL-2Rα (CD25): Specific for IL-2, binds with low affinity

• IL-2Rβ (CD122): Also a component of the IL-15 receptor, binds IL-2 with intermediate affinity

• Common gamma chain (γc/IL-2Rγ): Shared with receptors for IL-4, IL-7, IL-9, IL-15, and IL-21

Upon IL-2 binding, signal transduction occurs primarily through the IL-2Rβ and γc components, activating three major pathways :

• JAK/STAT pathway:

  • JAK1 and JAK3 phosphorylate STAT5

  • Phosphorylated STAT5 dimerizes and translocates to the nucleus

  • Activates transcription of target genes including CD25, creating a positive feedback loop

• PI3K/AKT pathway:

  • Promotes cell survival and metabolic reprogramming

  • Enhances glucose uptake and glycolysis in activated T cells

• RAS/MAPK pathway:

  • Drives cell cycle progression and proliferation

  • Contributes to cytokine production

Key observable effects in experimental systems include:

• Robust proliferation of activated T cells

• Protection from activation-induced cell death

• Enhanced expression of effector cytokines in CD8+ T cells

• Promotion of regulatory T cell development and maintenance

• Inhibition of Th17 cell differentiation

Researchers should note that IL-2 effects are context-dependent, varying with T cell subset, activation state, and concurrent signals from other cytokines and receptors.

How can researchers effectively use mouse IL-2 to study the balance between effector and regulatory T cell responses?

IL-2 plays a dual role in the immune system, promoting both effector T cell responses and regulatory T cell (Treg) development, making it an excellent tool for studying immune balance:

Experimental approaches for studying this balance:

• Dose-dependent effects:

  • Low IL-2 doses (0.1-1 ng/mL) preferentially support Treg maintenance due to their constitutively high expression of CD25 (IL-2Rα)

  • Higher doses (5-20 ng/mL) activate both Tregs and conventional T cells

  • Researchers can exploit this differential sensitivity to selectively manipulate Treg versus effector responses

• Timing considerations:

  • Early IL-2 addition during T cell activation promotes effector differentiation

  • IL-2 exposure during later phases can expand established Tregs

  • Time-course experiments with IL-2 addition/blocking can reveal critical windows for effector versus regulatory balance

• In vitro suppression assays:

  • Co-culture responder T cells with varying ratios of Tregs

  • Manipulate IL-2 concentrations to assess its role in suppressive function

  • Use anti-IL-2 or anti-IL-2R antibodies to block endogenous IL-2 signaling

• Analysis markers for distinguishing populations:

  • Tregs: CD4+CD25+Foxp3+

  • Effector T cells: CD25+, but Foxp3-, with subset-specific cytokine production

IL-2 contributes to T cell homeostasis in complex ways, promoting the Fas-induced death of naïve CD4+ T cells while sparing activated CD4+ memory lymphocytes . This highlights its central role in natural suppression of autoimmunity through Treg maintenance . Researchers studying autoimmune diseases or cancer immunotherapy should carefully consider how IL-2 manipulation affects this critical balance.

What methodological approaches can researchers use to investigate IL-2-dependent cell signaling networks?

Studying IL-2-dependent signaling networks requires multiple complementary methodological approaches:

• Phosphoprotein analysis:

  • Western blotting for phosphorylated forms of STAT5, AKT, and ERK following IL-2 stimulation

  • Phospho-flow cytometry for single-cell resolution of pathway activation

  • Time-course experiments to capture transient versus sustained signaling events

• Transcriptional profiling:

  • RNA-seq or microarray analysis of IL-2-stimulated cells at different time points

  • ChIP-seq for STAT5 binding sites to identify direct IL-2 target genes

  • Single-cell RNA-seq to distinguish heterogeneous responses within populations

• Functional signaling analysis:

  • Specific pathway inhibitors (JAK inhibitors, PI3K inhibitors, MEK inhibitors) to dissect contribution of each pathway

  • CRISPR/Cas9-mediated knockout of pathway components

  • Expression of constitutively active or dominant negative signaling proteins

• Receptor dynamics analysis:

  • Flow cytometry for surface expression of IL-2R components

  • Imaging techniques to visualize receptor clustering and internalization

  • Proximity ligation assays to detect receptor complex formation

• In vivo approaches:

  • Conditional knockout models for IL-2 or IL-2R components

  • Adoptive transfer of cells with reporter constructs for pathway activation

  • Treatment with recombinant IL-2 or IL-2/antibody complexes that selectively target different cell populations

When designing these experiments, researchers should carefully consider cellular activation state, timing of IL-2 addition, and the presence of other cytokines that may synergize with or antagonize IL-2 signaling, as these factors significantly impact experimental outcomes and interpretation.

What purity and endotoxin standards should researchers expect for recombinant mouse IL-2, and how do these factors impact experimental outcomes?

High-quality recombinant mouse IL-2 should meet specific purity and endotoxin standards to ensure reliable experimental results:

Purity standards:

• Commercial recombinant mouse IL-2 should be ≥95% pure as determined by SDS-PAGE analysis

• Purity is typically verified by silver staining, showing a band at approximately 17-19 kDa

• Additional verification methods include absorbance assays based on the Beers-Lambert law

Endotoxin standards:

• Endotoxin contamination should be ≤0.1 ng/μg (0.1 EU/μg) of mouse IL-2

• Testing is typically performed using a chromogenic Limulus Amebocyte Lysate (LAL) assay

Impact on experimental outcomes:

• Protein contaminants:

  • May provide unintended stimulation of immune cells

  • Can introduce confounding biological activities

  • May interfere with quantitative measurements

• Endotoxin contamination:

  • Activates TLR4 signaling, leading to pro-inflammatory cytokine production

  • Can cause aberrant activation of dendritic cells, macrophages, and B cells

  • May synergize with IL-2 to alter T cell differentiation patterns

  • Critical concern for in vivo studies due to potential systemic inflammation

• Host cell proteins:

  • Expression system-derived proteins may have immunomodulatory effects

  • Important consideration when switching between E. coli and yeast-derived products

Researchers should carefully review product specifications and consider additional quality control testing when inconsistent results are observed or when transitioning to new IL-2 preparations. For particularly sensitive applications, such as in vivo studies or work with primary immune cells, selecting preparations with documented low endotoxin levels and high purity is essential.

How can researchers design proper controls when using recombinant mouse IL-2 in complex immunological assays?

Designing appropriate controls is critical for interpreting results from experiments using recombinant mouse IL-2:

Essential controls for IL-2 experiments:

• Vehicle control:

  • Include a control containing all components used in IL-2 reconstitution (PBS, carrier protein, etc.)

  • Essential for distinguishing IL-2-specific effects from carrier effects

  • Particularly important when using preparations containing BSA or other carriers

• Dose-response controls:

  • Include multiple IL-2 concentrations spanning sub-optimal to saturating levels

  • Helps identify threshold effects and establish dose-dependent relationships

  • Typically should include concentrations ranging from 0.1-100 ng/mL

• Timing controls:

  • Include conditions where IL-2 is added at different time points

  • Critical for determining temporal requirements for IL-2 signaling

  • Helps distinguish between early and late effects of IL-2

• Blocking controls:

  • Anti-IL-2 or anti-IL-2R antibodies to confirm specificity of observed effects

  • Isotype control antibodies to account for non-specific antibody effects

  • IL-2 receptor knockout or knockdown cells where feasible

• Alternative cytokine controls:

  • Include related cytokines (IL-15, IL-7) that share receptor components

  • Helps distinguish IL-2-specific effects from general γc cytokine effects

  • Consider IL-2 mutants with altered receptor binding properties

• Biological activity verification:

  • Include a standardized CTLL-2 proliferation assay to confirm IL-2 bioactivity

  • Essential when troubleshooting unexpected results or using new IL-2 preparations

These controls should be tailored to the specific experimental question and cell types under investigation. For complex immunological assays involving multiple cell types, researchers should consider how IL-2 might affect each population differently and design controls accordingly.

How can researchers optimize the use of recombinant mouse IL-2 for expanding regulatory T cells ex vivo?

Ex vivo expansion of regulatory T cells (Tregs) using recombinant mouse IL-2 requires specific optimization strategies:

Isolation and culture conditions:

• Starting population:

  • FACS-sort CD4+CD25highFoxp3+ cells (if using Foxp3 reporter mice)

  • Alternatively, isolate CD4+CD25high cells using magnetic separation

  • Purity assessment is critical as conventional T cell contamination can outgrow Tregs

• Base medium and supplements:

  • Complete RPMI-1640 or IMDM medium

  • 10% heat-inactivated FBS (low endotoxin grade)

  • 2 mM L-glutamine, non-essential amino acids, sodium pyruvate

  • 50 μM β-mercaptoethanol (critical for murine T cells)

• IL-2 concentration optimization:

  • High IL-2 concentrations (100-1000 IU/mL or 10-100 ng/mL) are typically required

  • Add fresh IL-2 every 2-3 days due to consumption and degradation

  • Monitor Foxp3 expression regularly as IL-2 withdrawal can lead to instability

• TCR stimulation:

  • Initial stimulation with plate-bound anti-CD3 (1-5 μg/mL) and soluble anti-CD28 (1-2 μg/mL)

  • Alternatively, use anti-CD3/CD28 coated beads at 1:1 to 3:1 bead-to-cell ratio

  • Restimulation every 10-14 days may be required for continued expansion

• Additional cytokines to consider:

  • TGF-β (2-5 ng/mL) helps maintain Foxp3 expression

  • Rapamycin (100 nM) selectively inhibits conventional T cell outgrowth

  • IL-2/anti-IL-2 antibody complexes can enhance Treg selectivity

• Monitoring parameters:

  • Track proliferation by cell counting or dye dilution

  • Assess Foxp3 stability by flow cytometry

  • Verify suppressive function in standard suppression assays

Optimization should involve systematic testing of these parameters with careful documentation of Treg phenotype stability and suppressive function. The optimal protocol may vary depending on the mouse strain and the experimental application for which the expanded Tregs will be used.

What are the key considerations when designing experiments to investigate the role of IL-2 in memory T cell formation and maintenance?

Investigating IL-2's role in memory T cell biology requires careful experimental design:

Key experimental considerations:

• Timing of IL-2 manipulation:

  • IL-2 signals during primary response affect memory cell programming

  • Early IL-2 (days 0-3 post-activation) influences effector differentiation

  • Later IL-2 (days 4-8) may affect memory precursor formation

  • Long-term IL-2 availability impacts memory cell maintenance

• Memory T cell subsets:

  • Central memory (TCM): CD44high CD62Lhigh CCR7+

  • Effector memory (TEM): CD44high CD62Llow CCR7-

  • Tissue-resident memory (TRM): CD44high CD69+ CD103+

  • IL-2 may differentially affect these populations

• Experimental approaches:

  • In vitro models:

    • Two-phase culture systems (activation phase followed by rest phase)

    • Varying IL-2 concentrations during different phases

    • Measuring phenotypic markers, recall responses, and metabolism

  • In vivo models:

    • Adoptive transfer of TCR-transgenic T cells followed by antigen challenge

    • Neutralizing IL-2 or blocking IL-2R at different timepoints

    • IL-2/anti-IL-2 complexes for selective targeting of IL-2 to different cell populations

• Functional readouts:

  • Proliferative recall response to antigen restimulation

  • Cytokine production profile (IFN-γ, TNF-α, IL-2)

  • Expression of transcription factors associated with memory (Eomes, Bcl-6, TCF-1)

  • Metabolic profile (mitochondrial mass, spare respiratory capacity)

• Temporal considerations:

  • Short-term memory (2-4 weeks post-priming)

  • Long-term memory (8+ weeks post-priming)

  • Secondary and tertiary responses to assess recall quality

IL-2 is particularly important for CD8+ T cell memory formation, where it helps program the development of functional memory precursors during the primary response. For comprehensive analysis, researchers should combine phenotypic characterization with functional assessments and consider both quantity and quality of the resulting memory populations.

How are engineered IL-2 variants being used to selectively target specific cell populations in immunological research?

Engineered IL-2 variants represent an important frontier in IL-2 research, offering selective targeting capabilities:

Key engineered IL-2 approaches:

• IL-2/anti-IL-2 antibody complexes:

  • JES6-1 mAb (anti-IL-2) + IL-2: Preferentially targets CD25high cells (Tregs)

  • S4B6 mAb (anti-IL-2) + IL-2: Preferentially targets CD122high cells (CD8+ T cells, NK cells)

  • These complexes extend IL-2 half-life and direct activity to specific cell populations

  • Enable selective expansion of Tregs or effector cells in vivo

• IL-2 muteins with altered receptor binding:

  • Mutations at the IL-2Rα (CD25) binding site: Reduce Treg targeting

  • Mutations at the IL-2Rβ (CD122) interface: Alter signaling strength

  • "Super-2" variants: Enhanced binding to IL-2Rβ without requiring CD25 co-expression

• Receptor-selective IL-2 fusion proteins:

  • IL-2 fused to antibody fragments targeting specific cell markers

  • Cytokine-cytokine fusions (e.g., IL-2-IL-15 or IL-2-IL-33 chimeras)

  • PEGylated variants with altered pharmacokinetics and distribution

• Applications in mouse research models:

  • Autoimmunity: Treg-selective IL-2 complexes (JES6-1/IL-2) to suppress inflammation

  • Cancer immunotherapy: CD8+ T cell-selective complexes (S4B6/IL-2) to enhance anti-tumor responses

  • Infectious disease: Engineered IL-2 variants to boost pathogen-specific T cell responses

  • Transplantation: Treg-selective IL-2 to promote tolerance

These approaches allow researchers to dissect the cell type-specific roles of IL-2 signaling with greater precision than possible with conventional recombinant IL-2. When designing experiments with these tools, researchers should include appropriate controls to verify the selective targeting of intended cell populations and confirm that the engineered variants maintain the core signaling properties of wild-type IL-2.

What emerging technologies are enhancing our understanding of IL-2 biology in mouse models?

Several cutting-edge technologies are transforming IL-2 research in mouse models:

• Single-cell technologies:

  • scRNA-seq reveals heterogeneous responses to IL-2 within seemingly uniform populations

  • CITE-seq (cellular indexing of transcriptomes and epitopes) enables simultaneous measurement of IL-2 receptor components and downstream gene expression

  • Single-cell ATAC-seq identifies chromatin accessibility changes induced by IL-2 signaling

• Advanced imaging approaches:

  • Intravital microscopy to visualize IL-2 production and consumption in vivo

  • IL-2 reporter mice that express fluorescent proteins under IL-2 or IL-2R promoters

  • Multiphoton imaging of T cell zones to study IL-2 gradient formation and cellular interactions

• Genetic engineering advances:

  • CRISPR/Cas9-mediated precise genome editing of IL-2 and IL-2R genes

  • Conditional and inducible knockout/knockin models for temporal control

  • Tissue-specific IL-2 or IL-2R deletion to study compartmentalized effects

• Structural biology insights:

  • Cryo-EM structures of the IL-2/IL-2R complex revealing molecular interaction details

  • Structure-guided design of IL-2 variants with altered receptor binding properties

  • Molecular dynamics simulations predicting functional consequences of IL-2 mutations

• Advanced mouse models:

  • Humanized mouse models expressing human IL-2 and IL-2R components

  • Reporter systems for IL-2 signaling pathway activation (e.g., STAT5 translocation)

  • Models with fluorescently tagged endogenous IL-2 to track its production and distribution

These technologies are enabling researchers to address previously intractable questions about the spatiotemporal dynamics of IL-2 signaling, cell-specific responses, and the formation of IL-2 niches within tissues. When incorporating these approaches, researchers should consider the technical limitations and validation requirements specific to each technology.