Recombinant Mouse C-X-C motif chemokine 14 protein (Cxcl14) (Active)

What is the molecular structure of recombinant mouse CXCL14?

Recombinant mouse CXCL14 is a full-length mature protein consisting of 77 amino acid residues (expression range 23-99aa). The complete amino acid sequence is: SKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIVTTKSMSRYRGQEHCLHPKLQSTKRFIKWYNAWNEKRRVYEE . The protein has a molecular weight of approximately 9.4 kDa in tag-free form or 13.4 kDa with N-terminal His-tag . The protein belongs to the intercrine alpha (chemokine CXC) family and lacks the characteristic ELR domain preceding the CXC motif that is found in other CXC chemokines .

How is purity and endotoxin level determined for research-grade recombinant mouse CXCL14?

High-quality recombinant mouse CXCL14 typically demonstrates >90-95% purity as determined by SDS-PAGE and HPLC analysis . Endotoxin contamination is evaluated using the LAL (Limulus Amebocyte Lysate) method, with acceptable levels being less than 1.0 EU/μg for most experimental applications . These parameters are critical for ensuring experimental reproducibility and preventing endotoxin-mediated cellular responses that could confound research findings.

What is the bioactivity profile of recombinant mouse CXCL14?

The biological activity of recombinant mouse CXCL14 is typically determined through chemotaxis bioassays using human monocytes. Fully active preparations demonstrate chemotactic activity within a concentration range of 1.0-10 ng/ml . The protein serves as a chemoattractant for CESS B-cells and THP-1 monocytes, but not T-cells . It also functions as a chemoattractant for activated macrophages, immature dendritic cells, and natural killer cells, while exhibiting antiangiogenic properties by preventing endothelial cell migration .

What are the recommended storage conditions for maintaining CXCL14 stability?

For long-term storage, recombinant mouse CXCL14 should be stored at -20°C/-80°C, with -80°C being optimal for extended periods. The protein is typically available in either liquid or lyophilized powder form. For liquid preparations, addition of 5-50% glycerol (with 50% being standard) is recommended before aliquoting to prevent freeze-thaw damage . Working aliquots can be stored at 4°C for up to one week. Protein in liquid form generally remains stable for up to 6 months at -20°C/-80°C, while lyophilized powder maintains stability for up to 12 months .

What is the proper protocol for reconstituting lyophilized recombinant mouse CXCL14?

The recommended reconstitution protocol involves:

• Briefly centrifuging the vial prior to opening to bring contents to the bottom

• Reconstituting in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Adding glycerol to a final concentration of 5-50% for storage

• Creating multiple small aliquots to avoid repeated freeze-thaw cycles

For lyophilized preparations, the buffer before lyophilization typically consists of a Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain protein integrity during the freeze-drying process .

How can recombinant CXCL14 be utilized to study leukemic stem cell biology?

Recent research demonstrates that CXCL14 plays a critical role in chronic myeloid leukemia (CML) biology. Methodological approaches include:

• Co-culture systems: CML CD34+CD38- cells can be co-cultured with CXCL14-overexpressing stromal cells (NIH3-CXCL14) or control stromal cells (NIH3-CTRL) to evaluate the impact on leukemic stem cell (LSC) maintenance and differentiation. Measure outcomes using:

  • Long-term culture-initiating cell (LTC-IC) assays to assess stemness

  • Flow cytometry to quantify CD34+CD38- LSC frequency and CD15+CD66B+ myeloid cell differentiation

  • In vivo transplantation into immunodeficient mice to evaluate engraftment capacity

• Combination therapy assessment: Recombinant CXCL14 (alone or in combination with tyrosine kinase inhibitors like imatinib) can be tested in co-culture systems with normal or CML-derived bone marrow mesenchymal stromal cells (MSCs) to evaluate combinatorial effects on LSC survival .

Results from these approaches have shown that CXCL14 promotes proliferation and differentiation of CML LSCs while significantly reducing their engraftment potential in xenotransplantation models, suggesting CXCL14 as a potential therapeutic target .

Table based on data from Figure 4B in reference

What are the molecular mechanisms underlying CXCL14's anti-leukemic effects?

RNA sequencing analysis of CML CD34+CD38- cells treated with CXCL14 reveals several important molecular pathways affected:

• Downregulation of mTORC1 signaling and oxidative phosphorylation (OXPHOS): Key genes like OPA3, CYC1, and ATP2A2, important for cell energy metabolism and growth, are markedly reduced after CXCL14 treatment .

• Reduction in downstream oncogenic pathways: MYC targets, E2F targets, and G2M checkpoints that are downstream of mTORC1/OXPHOS and BCR-ABL1 activation are downregulated .

• Upregulation of TNF-α and TGF-β signaling: These pathways are enhanced following CXCL14 stimulation .

• Loss of leukemia-specific markers: IL1RAP, a marker specific for CML LSCs, is lost after CXCL14 stimulation, suggesting selective targeting of leukemic stem cells .

• Effects on mitochondrial function: CXCL14 reduces CYC1 protein levels, decreases reactive oxygen species production, and impairs mitochondrial membrane potential after the addition of pyruvate/malate, indicating compromised mitochondrial complex I activity .

These findings provide potential methodological approaches for investigating CXCL14's effects on cellular metabolism and mitochondrial function in other experimental systems.

What experimental models are appropriate for investigating CXCL14's immunomodulatory properties?

Several experimental models have been developed and validated for studying CXCL14's role in immune regulation:

• Transgenic mouse models: CXCL14 transgenic mice can be used to evaluate the protein's effects in various disease contexts. Studies show these mice exhibit suppressed carcinogenesis rates, decreased tumor volume, reduced pulmonary metastasis, and increased survival following tumor cell injection .

• In vitro chemotaxis assays: These assays quantitatively measure the chemotactic activity of CXCL14 on various immune cell populations. Cell types shown to respond to CXCL14 include:

  • Activated macrophages

  • Immature dendritic cells

  • Natural killer cells

  • Prostaglandin E2 or forskolin-treated monocytes (highly selective response)

• CXCL12/CXCR4 signaling modulation assays: Methods to evaluate CXCL14's inhibitory effect on the CXCL12/CXCR4 signaling pathway, which influences T-helper cell polarization. CXCL14 has been shown to promote Th1 immune responses under both physiological and pathological conditions .

How should researchers approach conflicting data regarding CXCL14's roles in different tissue contexts?

When addressing contradictory findings on CXCL14 function, researchers should consider several methodological factors:

• Dose-dependent effects: At physiological concentrations found in CXCL14 transgenic mice, the protein exhibits anti-tumor effects, while at higher concentrations (100-300 times higher) used in some in vitro studies, it may bind to CXCR4 and modulate receptor structure to enhance CXCL12 binding and activity .

• Cellular context: NIH-3T3 cells (commonly used as they readily incorporate foreign genes) may produce higher CXCL14 concentrations than physiologically relevant, potentially explaining pro-tumorigenic and angiogenic effects observed in some studies .

• Co-presence of other factors: The presence of molecules like CXCL12 may influence experimental outcomes and explain seemingly contradictory results .

• Tissue-specific expression patterns: CXCL14 shows varying expression levels across tissues: high in brain, lung, ovary, muscle, kidney, and liver parenchyma, with lower levels in bone marrow . These differences may contribute to context-dependent functions.

• Species-specific differences: Despite structural homology and similarity in tissue distribution, human and murine CXCL14 may have distinct species-specific functions in epithelial immunity .

What techniques are recommended for investigating CXCL14's role in central nervous system function?

Research into CXCL14's functions in the nervous system should consider:

• Temporal expression analysis: Studies have identified transient expression of CXCL14 by Purkinje cells in the developing cerebellum, suggesting involvement in postnatal cerebellar maturation .

• Behavioral assays: Evidence indicates CXCL14 may play an important role in central nervous system regulation of feeding behavior .

• Developmental timing: When studying neurological effects, the developmental stage is critical as CXCL14 may have stage-specific functions during brain development.

What are appropriate methodologies for studying CXCL14 in metabolic disease models?

For metabolic research, the following approaches have proven informative:

• Diet-induced obesity models: CXCL14 plays a causal role in high-fat diet-induced obesity, making this a valuable model system .

• Macrophage recruitment assays: CXCL14 functions as a critical chemoattractant of white adipose tissue macrophages, suggesting a role in obesity-associated inflammation .

• Glucose metabolism studies: CXCL14 regulates glucose metabolism primarily in skeletal muscle, indicating tissue-specific metabolic effects that should be evaluated in relevant tissue models .

How should researchers approach evaluating CXCL14's role in vascular biology?

When investigating CXCL14 in vascular contexts:

• Platelet isolation and activation studies: Platelets are a relevant source of CXCL14, and platelet-derived CXCL14 at vascular lesion sites may play important roles in vascular repair and regeneration .

• Endothelial migration assays: CXCL14 functions as an antiangiogenic factor by preventing endothelial cell migration, making this a key functional readout .

• Vascular repair models: In vivo models of vascular injury can help elucidate CXCL14's role in repair processes.

What experimental approaches are recommended for studying CXCL14 in reproductive biology?

Research on CXCL14's role in reproductive biology should consider:

• Uterine NK cell studies: CXCL14 appears to have important functions in uterine NK (uNK) cells, and proper CXCL14 protein levels are required to recruit NK cells to the pregnant uterus .

• Trophoblast outgrowth models: CXCL14 functions as an important paracrine/autocrine modulator regulating trophoblast outgrowth at the maternal-fetal interface during pregnancy establishment .

• Quantitative analysis of CXCL14 levels: Ensuring appropriate protein concentration is critical, as both excessive and insufficient levels may disrupt normal reproductive processes.

What are the critical quality control parameters for recombinant CXCL14 in experimental systems?

When utilizing recombinant mouse CXCL14, researchers should verify:

• Protein purity: Greater than 90-95% as determined by SDS-PAGE and HPLC

• Endotoxin levels: Less than 1.0 EU/μg as determined by the LAL method

• Biological activity: Confirmation via chemotaxis bioassays using appropriate target cells

• Proper protein folding: Especially important when using E. coli-derived recombinant proteins

• Batch-to-batch consistency: Testing multiple parameters across different production lots

How can researchers address the lack of identified specific receptors for CXCL14?

Despite extensive research, specific CXCL14 receptors remain incompletely characterized. Methodological approaches to address this challenge include:

• Receptor binding assays: Using labeled recombinant CXCL14 to identify potential binding partners on responsive cell types

• Signaling pathway inhibition studies: Systematic inhibition of various signaling components to identify downstream pathways

• Cross-linking experiments: To capture transient protein-protein interactions

• Competition assays: With known chemokines to identify shared receptor usage

• High-throughput screening: Of potential G-protein coupled receptors as candidate CXCL14 receptors

What emerging methodologies might advance our understanding of CXCL14 biology?

Several cutting-edge approaches hold promise for CXCL14 research:

• Single-cell RNA sequencing: To identify cell-specific responses to CXCL14 in heterogeneous tissues

• CRISPR/Cas9-mediated genome editing: For precise manipulation of CXCL14 and potential receptor genes

• Advanced imaging techniques: Such as intravital microscopy to observe CXCL14-mediated cell migration in vivo

• Proteomics approaches: To identify protein interaction networks influenced by CXCL14

• Computational modeling: Of CXCL14's structural interactions with potential binding partners

How might researchers resolve the apparent context-dependent functions of CXCL14 in cancer?

To address the seemingly contradictory roles of CXCL14 in cancer:

• Systematic concentration-response studies: Testing physiologically relevant concentration ranges

• Context-specific experimental designs: Comparing effects in multiple cancer types and microenvironments

• Co-factor analysis: Identifying molecules that modify CXCL14 function in different contexts

• Temporal expression studies: Investigating how CXCL14's effects may vary during different stages of cancer progression

• Combined in vitro and in vivo approaches: To validate findings across experimental systems