Recombinant Rat C-X-C motif chemokine 3 protein (Cxcl3) (Active)

What is Recombinant Rat CXCL3 and what are its primary functions in biological systems?

Recombinant Rat CXCL3, also known as Growth regulated oncogene-gamma (GRO gamma), belongs to the family of chemotactic cytokines called chemokines. It is identical to MGSA (melanoma growth stimulatory activity) and functions primarily as a potent neutrophil attractant and activator. As a ligand for CXCR2, CXCL3 exhibits significant chemotactic activity for neutrophils and may play crucial roles in inflammation processes . The protein exerts its effects on endothelial cells in an autocrine fashion and participates in immune response as both a signal and effector molecule. In inflammation models, CXCL3 facilitates neutrophil recruitment to sites of inflammation or infection, serving as an important mediator in the immune response cascade .

What are the common alternative names and identifiers for Rat CXCL3 in scientific literature?

When conducting literature searches for Rat CXCL3, researchers should be aware of its multiple designations:

• C-X-C motif chemokine 3

• Cytokine-induced neutrophil chemoattractant 2 (CINC-2)

• Macrophage inflammatory protein 2-alpha/beta (MIP2-alpha/beta)

• Growth regulated oncogene-gamma (GRO gamma)

• Gene Symbol: CXCL3

• Gene ID: 171551

• Accession: Q10746

These alternative nomenclatures are important when performing comprehensive literature reviews or designing experimental protocols, as different research groups may use varying terminology when referring to this protein in publications.

What are the optimal methods for detecting and quantifying Rat CXCL3 in experimental samples?

For quantitative measurement of Rat CXCL3, ELISA represents the gold standard method. Single-wash 90-minute SimpleStep ELISA® kits provide efficient quantification in various sample types including heparin plasma, citrate plasma, cell culture supernatant, serum, and EDTA plasma samples . These assays employ a sandwich ELISA approach allowing the formation of the antibody-analyte sandwich complex in a single step, significantly reducing assay time compared to traditional methods.

When choosing between different detection methods, researchers should consider the following performance characteristics:

Immunohistochemistry represents another valuable method for tissue-based detection, using antibodies targeted to CXCL3 (typically at 1:100 dilution), followed by treatment with secondary detection systems such as SABC Kit and visualization with 3,3′-diaminobenzidine (DAB) .

How should researchers prepare and handle recombinant Rat CXCL3 for maximum stability and activity?

Recombinant Rat CXCL3 is typically supplied as a lyophilized powder from a 0.2 μm filtered solution in PBS . To ensure optimal stability and activity:

• Perform a quick spin of the vial followed by reconstitution in distilled water to a concentration not less than 0.1 mg/mL

• This solution can then be diluted into other buffers as needed for specific experiments

• For long-term storage, maintain aliquots at -20°C or -80°C to prevent repeated freeze-thaw cycles

• Verify activity before use, especially after extended storage periods

• When designing experiments, account for the molecular weight of approximately 8 kDa when calculating molar concentrations

The ED(50) for CXCL3, determined by the dose-dependent proliferation of HepG2 cells, is typically less than 0.5ng/mL, which should be considered when designing concentration-response studies .

How can recombinant Rat CXCL3 be used to study cancer progression mechanisms?

Recent research has demonstrated significant correlations between CXCL3 expression and cancer progression, particularly in colorectal cancer models. To investigate CXCL3's role in cancer:

• Exogenous administration approach: Culture cancer cell lines (such as HT-29 or SW480) with different concentrations of recombinant CXCL3 to assess proliferation using CCK-8 assays, migration using Transwell systems, and colony formation abilities .

• Genetic modulation approach: Establish CXCL3-overexpressing or CXCL3-deficient cell lines through transfection with lentiviral expression vectors containing CXCL3 gene sequences or interfering sequences targeted at CXCL3 .

• Signaling pathway analysis: Evaluate the effects of CXCL3 on key signaling pathways (particularly the ERK pathway) by monitoring the expression of ERK, p-ERK1/2, Bcl-2, Cyclin D1, and Bax using western blotting assays. This approach has revealed that CXCL3 overexpression increases ERK, p-ERK1/2, Bcl-2, and Cyclin D1 protein levels in certain cancer cell lines .

• Pathway inhibition studies: Use specific inhibitors such as the ERK1/2 blocker PD98059 to determine whether CXCL3's effects are mediated through specific pathways .

What considerations should be made when studying CXCL3 interactions with CXCR2 receptors?

When investigating CXCL3-CXCR2 interactions, researchers should consider:

• Receptor specificity: While CXCL3 is primarily a ligand for CXCR2, potential cross-reactivity with other chemokine receptors should be assessed in the experimental system .

• Receptor expression profiling: Prior to conducting binding studies, verify the expression levels of CXCR2 on target cells using flow cytometry or other appropriate methods.

• Competition studies: Design experiments that account for potential competition with other alpha chemokines that also bind to CXCR2.

• High-affinity binding to IL-8 receptor type B: CXCL3, like other GRO proteins, can bind with high affinity to the IL-8 receptor type B, which should be considered when interpreting results of binding and functional studies .

• Glycosaminoglycan (GAG) interactions: Consider that CXCL3, as a basic protein, likely binds avidly to negatively charged GAG molecules both on cell surfaces and in the extracellular matrix, which can affect its bioavailability and function .

What controls should be included when studying CXCL3-mediated effects in cell culture systems?

A robust experimental design for studying CXCL3-mediated effects should include:

• Negative controls:

  • Vehicle control (buffer without CXCL3)

  • Mock-transfected cells for gene expression studies

  • Isotype control antibodies for neutralization studies

• Positive controls:

  • Known CXCR2 agonists (other than CXCL3)

  • Positive readouts for the specific assay being employed

• Dosage controls:

  • Concentration-response experiments (typically ranging from 0.1-100 ng/mL)

  • Time-course analyses to determine optimal incubation periods

• Specificity controls:

  • CXCR2 receptor antagonists to confirm receptor-dependent effects

  • CXCL3 neutralizing antibodies to confirm ligand specificity

  • Pathway inhibitors (e.g., PD98059 for ERK pathway) to confirm downstream signaling

• Technical replicates:

  • At minimum, experiments should be performed in triplicate to ensure statistical validity

How should researchers differentiate between the roles of CXCL3 and related chemokines (CXCL9, CXCL10, CXCL11) in experimental systems?

Differentiating between the specific contributions of CXCL3 and related chemokines requires careful experimental design:

• Expression profiling:

  • Analyze the expression patterns of multiple chemokines simultaneously under various stimulation conditions

  • Monitor induction by different stimuli, as CXCL9 is primarily induced by IFN-γ, CXCL10 by IFN-γ>IFN-α/β>TNF, and CXCL11 by IFN-γ=IFN-β>IFN-α>TNF

• Receptor binding analysis:

  • Perform competitive binding assays to determine receptor preference and binding affinities

  • Remember that CXCL3 primarily signals through CXCR2, while CXCL9, CXCL10, and CXCL11 activate CXCR3

• Selective inhibition:

  • Use specific neutralizing antibodies against individual chemokines

  • Employ siRNA or CRISPR-Cas9 approaches for selective gene knockdown/knockout

• Functional redundancy assessment:

  • Design experiments that can detect additive, synergistic, or antagonistic effects between these chemokines

  • Consider that these chemokines can have redundant functions but may also collaborate or compete with each other

What are common challenges when working with recombinant CXCL3 and how can they be addressed?

Researchers frequently encounter several challenges when working with recombinant CXCL3:

• Protein aggregation:

  • Issue: CXCL3 may form aggregates during storage or experimental procedures

  • Solution: Include low concentrations (0.1-0.5%) of carrier proteins such as BSA in buffers; avoid repeated freeze-thaw cycles; centrifuge briefly before use to remove any aggregates

• Loss of activity:

  • Issue: Decreased biological activity after storage

  • Solution: Store as small aliquots at -80°C; validate activity periodically using established functional assays such as chemotaxis or HepG2 proliferation assays

• Non-specific binding:

  • Issue: High background in binding assays due to CXCL3's affinity for GAGs and other charged molecules

  • Solution: Include appropriate blocking agents and consider pre-incubation steps to reduce non-specific interactions

• Variable cell responses:

  • Issue: Inconsistent cellular responses to CXCL3 stimulation

  • Solution: Verify receptor expression levels; ensure consistent cell culture conditions; validate the recombinant protein's activity before use

• Detection limitations in complex samples:

  • Issue: Difficulty in detecting endogenous CXCL3 in biological samples

  • Solution: Optimize sample preparation procedures; consider concentration steps; use high-sensitivity ELISA kits with validated recovery rates for specific sample types

How can researchers effectively control for batch-to-batch variability in recombinant CXCL3 experiments?

To control for batch-to-batch variability:

• Activity normalization:

  • Characterize each new batch using standardized bioassays (e.g., chemotaxis or proliferation assays)

  • Calculate and use equi-active concentrations rather than relying solely on protein concentration

• Internal standards:

  • Maintain a reference standard from a well-characterized batch

  • Include this reference in each experiment for direct comparison

• Quality control metrics:

  • Verify purity (>95% as determined by SDS-PAGE)

  • Confirm endotoxin levels (<1.0 EU/μg of recombinant protein as determined by the LAL method)

  • Validate protein identity using mass spectrometry or other analytical techniques

• Experimental design adaptation:

  • When changing batches, perform side-by-side experiments with both old and new batches

  • Consider including batch as a factor in statistical analyses

How is CXCL3 being investigated in cancer research models, and what are the emerging therapeutic implications?

Current cancer research has highlighted CXCL3's potential role in tumorigenesis and progression:

• Expression correlation with clinical parameters:

  • Research has demonstrated that CXCL3 mRNA is significantly upregulated in colon adenocarcinoma (COAD) tissues compared to normal colon tissue

  • High expression levels correlate with clinical stage, race, gender, age, histological subtype, nodal metastasis, and TP53 mutation status

  • ROC curve analysis (AUC = 0.924, P < 0.0001) indicates that CXCL3 expression has high accuracy in clinical diagnosis of COAD

• Functional studies in cancer models:

  • Exogenous administration or overexpression of CXCL3 enhances malignant behaviors in cancer cell lines

  • CXCL3 deficiency inhibits cell proliferation, migration, and colony formation abilities in HT-29 and SW480 cells

  • These effects appear to be mediated through regulation of the ERK signaling pathway, with CXCL3 overexpression increasing ERK, p-ERK1/2, Bcl-2, and Cyclin D1 protein levels

• Therapeutic targeting approaches:

  • Inhibition of CXCL3-CXCR2 signaling using receptor antagonists

  • Neutralization of secreted CXCL3 using specific antibodies

  • Interference with downstream signaling pathways (particularly ERK1/2) using inhibitors such as PD98059

  • Gene silencing approaches targeting CXCL3 expression

• Biomarker potential:

  • Evaluation of CXCL3 as a diagnostic or prognostic biomarker in various cancer types

  • Development and validation of CXCL3-based prognostic models

What are the current methodological approaches for studying CXCL3's role in inflammation and immune response?

Advanced methodological approaches for studying CXCL3 in inflammation include:

• In vivo inflammation models:

  • Neutrophil recruitment assays in various tissue compartments

  • Assessment of CXCL3's role in acute vs. chronic inflammation models

  • Comparative analysis with other inflammatory chemokines

• Immunocyte interaction studies:

  • Investigation of CXCL3's effects on various immune cell populations beyond neutrophils

  • Analysis of CXCL3-induced functional changes in target cells (respiratory burst, degranulation, cytokine production)

  • Examination of receptor downregulation and desensitization after CXCL3 exposure

• Signaling pathway dissection:

  • Detailed mapping of CXCL3-triggered intracellular signaling cascades

  • Phosphoproteomic analyses to identify novel signaling nodes

  • Systems biology approaches to model CXCL3 network interactions

• Expression regulation studies:

  • Analysis of transcriptional control mechanisms governing CXCL3 expression

  • Investigation of post-transcriptional regulation including mRNA stability and microRNA interactions

  • Epigenetic profiling of the CXCL3 locus under various inflammatory conditions

These methodological approaches continue to expand our understanding of CXCL3's multifaceted roles in both physiological and pathological processes, offering new avenues for therapeutic intervention in inflammatory diseases and cancer.