Interleukin 8 (IL-8), also known as CXCL8, is a member of the CXC chemokine family. It is secreted by a variety of cell types including monocytes, macrophages, T cells, neutrophils, fibroblasts, endothelial cells, and various tumor cell lines in response to inflammatory stimuli (IL-1, TNF, LPS) (1). It is also synthesized by endothelial cells, which store IL-8 in vesicles. IL-8 functions as a chemoattractant for neutrophils, basophils, and T cells, but not monocytes (2). It also plays a vital role in tumorigenesis, progression and metastasis, and accordingly the expression levels of IL-8 correlate with disease progression in several human carcinomas (3). IL-8 has the N-terminal tripeptide ELR motif, which is known to mediate IL-8 receptor binding and subsequent signal transduction, such as the TRAF6-NF-κB pathway. It can also signal through the CXCR1 and CXCR2 receptors. IL-8 is believed to play a role in the pathogenesis of cystic fibrosis (4).
Protein Details
Purity
>97% by SDS-PAGE and analyzed by silver stain.
Endotoxin Level
<0.1 EU/µg as determined by the LAL method
Biological Activity
The biological activity of Human IL-8 was determined by its induction of myeloperoxidase release from human neutrophils (Schröeder, J. et al., 1987, J. Immunol. 139:3474), or chemotaxis of hCXCR-2 transfected mouse BaF/3 cells. The expected ED<sub>50</sub> for these effects are typically 0.15 - 0.3 μg/ml, or 0.5 - 2.5 ng/ml, respectively.
The predicted molecular weight of Recombinant Human IL-8 is Mr 8 kDa.
Predicted Molecular Mass
8
Formulation
This recombinant protein was lyophilized from a 0.2 μm filtered solution in deionized water.
Storage and Stability
This lyophilized protein is stable for six to twelve months when stored desiccated at -20°C to -70°C. After aseptic reconstitution, this protein may be stored at 2°C to 8°C for one month or at -20°C to -70°C in a manual defrost freezer. Avoid Repeated Freeze Thaw Cycles. See Product Insert for exact lot specific storage instructions.
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Recombinant Human IL-8 (CXCL8) is widely used in research because it is a key chemokine involved in inflammation, immune cell recruitment, angiogenesis, tumor progression, and as a biomarker for various diseases. Its recombinant form allows for controlled, reproducible studies of its biological effects in vitro and in vivo.
Key reasons to use recombinant human IL-8 in research applications:
Immune Cell Recruitment: IL-8 is a potent chemoattractant for neutrophils, basophils, and T cells, making it essential for studying leukocyte migration, inflammation, and innate immune responses.
Inflammation Models: IL-8 plays a central role in inflammatory diseases such as rheumatoid arthritis and airway inflammation, making it valuable for modeling and dissecting inflammatory pathways.
Cancer Research: IL-8 promotes tumor progression by enhancing angiogenesis (formation of new blood vessels), epithelial-mesenchymal transition (EMT), metastasis, and recruitment of immunosuppressive cells to the tumor microenvironment. It is used to study mechanisms of tumor growth, metastasis, and resistance to therapy.
Biomarker Studies: IL-8 is considered a universal biomarker for various pathological conditions, including infections, cancer, and inflammatory diseases, due to its elevated levels in these states.
Hematopoiesis and Cell Differentiation: IL-8 modulates myelopoiesis (formation of myeloid cells) and affects the homing and proliferation of hematopoietic progenitors, making it useful for stem cell and hematopoietic research.
Viral Pathogenesis: IL-8 can stimulate HIV-1 replication in macrophages and T lymphocytes, providing a model to study viral-host interactions and potential therapeutic interventions.
Standardization and Reproducibility: Using recombinant IL-8 ensures batch-to-batch consistency and defined activity, which is critical for reproducible experimental results in cell-based assays, bioassays, and ELISA standards.
Typical applications include:
Cell migration and chemotaxis assays
In vitro inflammation and immune response models
Tumor cell invasion, angiogenesis, and EMT studies
Biomarker validation and quantification assays
Functional studies of cytokine signaling pathways
In summary, recombinant human IL-8 is a versatile tool for dissecting the molecular and cellular mechanisms of inflammation, immunity, cancer, and other disease processes, enabling precise and reproducible experimental design.
Yes, recombinant human IL-8 is commonly used as a standard for quantification or calibration in ELISA assays for IL-8. This is a standard practice in both commercial ELISA kits and custom assay development.
Key points and supporting details:
Recombinant IL-8 as Standard: Most commercial ELISA kits for human IL-8 use recombinant human IL-8 as the calibration standard to generate the standard curve for quantification. This allows for accurate quantification of IL-8 in biological samples by comparing sample absorbance to that of known concentrations of the recombinant standard.
Parallelism and Accuracy: Studies and kit validations show that recombinant IL-8 standards yield standard curves that are parallel to those obtained with natural IL-8, indicating that quantification is accurate for both recombinant and endogenous IL-8.
Calibration and Traceability: Some kits calibrate their recombinant IL-8 standards against international reference materials, such as the NIBSC/WHO International Standard 89/520, providing traceability and allowing for conversion between units (e.g., pg/mL to IU/mL).
Application Protocol: When using recombinant IL-8 as a standard, it is essential to:
Reconstitute and dilute the protein according to the ELISA kit or assay protocol.
Prepare a standard curve with serial dilutions covering the expected range of IL-8 concentrations in your samples.
Ensure the recombinant IL-8 used matches the isoform and sequence recognized by the antibodies in your assay (most kits use the 72 or 77/78 amino acid forms).
Best Practices:
Use the same buffer and matrix for standard dilutions as for your samples to minimize matrix effects.
Confirm that your recombinant IL-8 is of high purity and bioactivity, and ideally, that it is validated for ELISA use.
If using a custom or non-kit recombinant IL-8, verify that it is recognized by the capture and detection antibodies in your assay.
In summary: Recombinant human IL-8 is suitable and widely accepted as a standard for ELISA quantification, provided it is compatible with your assay’s antibodies and protocol. Always follow the specific instructions of your ELISA kit or validated in-house protocol for optimal results.
Recombinant Human IL-8 (CXCL8) has been validated for a wide range of applications in published research, primarily in bioassays, ELISA (as a standard), and various cell-based functional studies.
Key validated applications include:
Bioassays: Used to assess chemotactic activity, particularly for neutrophil migration and activation, as well as for studying the recruitment and function of other immune cells such as monocytes and myeloid-derived suppressor cells.
ELISA Standard: Employed as a quantitative standard in ELISA assays to measure IL-8 levels in biological samples.
Cell Culture and Differentiation Studies: Utilized to investigate effects on cell proliferation, differentiation, and signaling, including studies on endothelial cells, hematopoietic progenitors, and pluripotent stem cells.
Functional Assays: Validated for use in assays measuring cell migration, angiogenesis, and immune cell activation.
Western Blot, Immunohistochemistry, Immunofluorescence, Immunoprecipitation, Flow Cytometry: Recombinant IL-8 has been used as a positive control or for validation in these immunodetection techniques.
Assay Validation: Used to validate the specificity and sensitivity of various immunoassays and cell-based assays.
Representative published research applications:
Neutrophil chemotaxis and activation: IL-8 is routinely used to induce and measure neutrophil migration and degranulation in vitro.
Cancer biology: Studies have used recombinant IL-8 to investigate its role in tumor angiogenesis, metastasis, and modulation of the tumor microenvironment.
Inflammation and immune response: IL-8 has been validated in models of acute and chronic inflammation, including atherosclerosis, colitis, and autoimmune diseases.
Stem cell research: Used to study CXCR1/CXCR2 signaling in pluripotent stem cell proliferation and differentiation.
Angiogenesis assays: IL-8 is applied to endothelial cell cultures to assess proliferation, migration, and tube formation.
Hematopoietic cell mobilization: Validated in studies examining the mobilization of hematopoietic stem and progenitor cells.
ELISA standardization: Used as a reference standard for quantifying IL-8 in biological fluids.
These applications are supported by numerous peer-reviewed studies and product validation data, confirming the utility of recombinant human IL-8 in diverse experimental settings.
To reconstitute and prepare Recombinant Human IL-8 protein for cell culture experiments, dissolve the lyophilized protein in sterile phosphate-buffered saline (PBS), ideally containing at least 0.1% carrier protein such as human or bovine serum albumin (BSA), to a concentration of 100 μg/mL. For carrier-free preparations, sterile PBS alone is sufficient.
Step-by-step protocol:
Equilibrate materials: Bring the vial of lyophilized IL-8 and your chosen buffer (PBS or PBS + 0.1% BSA) to room temperature before opening.
Centrifuge vial: Briefly spin the vial in a microcentrifuge (20–30 seconds) to collect all powder at the bottom.
Add buffer: Add sterile PBS (with or without 0.1% BSA, depending on formulation) to achieve the desired concentration (commonly 100 μg/mL for stock solution).
Dissolve protein: Gently mix by pipetting up and down or by gentle swirling. Do not vortex or shake vigorously, as this may denature the protein.
Incubate: Allow the solution to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution. If visible flakes remain, continue gentle mixing for up to 2 hours.
Aliquot and storage: Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles. Store aliquots at −20 °C or −80 °C for long-term storage; at 2–8 °C for short-term use (up to 1 month). Avoid storing at concentrations above 1 mg/mL.
Working dilution: For cell culture experiments, dilute the stock solution in your cell culture medium to the desired final concentration (e.g., 0.5–2.5 ng/mL for chemotaxis assays).
Additional notes:
Always consult the Certificate of Analysis (CoA) or product datasheet for specific instructions regarding your batch.
Carrier protein (BSA or HSA) helps stabilize IL-8 and prevents adsorption to plasticware, especially at low concentrations.
Avoid repeated freeze-thaw cycles to preserve bioactivity.
Do not vortex or mix vigorously to prevent protein denaturation.
Summary Table:
Step
Buffer/Condition
Notes
Equilibrate
Room temperature
Vial and buffer
Centrifuge vial
—
20–30 sec, collect powder
Add buffer
PBS ± 0.1% BSA
100 μg/mL stock recommended
Dissolve protein
Gentle mixing, no vortex
15–30 min, up to 2 h if needed
Aliquot & storage
−20 °C or −80 °C
Avoid freeze-thaw cycles
Working dilution
Cell culture medium
0.5–2.5 ng/mL typical
This protocol ensures optimal recovery and bioactivity of recombinant human IL-8 for cell culture applications.
References & Citations
1. Baggiolini, M. et al. (1992) FEBS Lett. 307:97
2. Köhidai, L. et al. (1998) Cytokine 10:481
3. Yuan, A. et al. (2006) Front. Biosci. 10:853
4. Tabary, O. et al. (1998) Amer. J. Pathol. 153:921
Products are for research use only. Not for use in diagnostic or therapeutic procedures.
