Human Interleukin-8 (72AA) Recombinant

/, CXC chemokines/Human Interleukin-8 (72AA) Recombinant

accession P10145


Source Optimized DNA sequence encoding Human Interleukin-8 mature chain was expressed in Escherichia Coli.
Molecular weight Native human IL-8, generated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calculated molecular mass of approximately 8 kDa. Recombinant IL-8 is a monomer protein consisting of 72 amino acid residue subunits, and migrates as an approximately 8 kDa protein under reducing conditions in SDS-PAGE.
Purity >95%, as determined by SDS-PAGE and HPLC
Biological Activity Determined by its ability to chemoattract human peripheral blood neutrophils using a concentration range of.0-100.0 ng/ml.

Protein Sequence MTSKLAVALL AAFLISAALC EGAVLPRSAK ELRCQCIKTY SKPFHPKFIK ELRVIESGPH CANTEIIVKL SDGRELCLDP KENWVQRVVE KFLKRAENS
Endotoxin Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation Recombinant IL-8 was lyophilized from a.2 μm filtered PBS solution pH7.5 .
Reconstitution A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers
Storage The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.


Interactor P25024
Interactor P25025
Interactor Q16570
Biological Process Chemotaxis
Biological Process Inflammatory-response
Molecular function Cytokine

Methods

Downstream target genes of GLO1 and their clinical correlations.

CXCL8
  • HIF-1α, NF-kB, VEGF, CXCL8, CXCL1, and CXCR2 protein levels in TSGH cells transfected with GLO1 shRNA (KG1 and KG2) and control shRNA (C1 and C2).

Enzyme-Linked Immunosorbent Assay (ELISA)

  • An ELISA kit was used according to the manufacturer's protocol to detect IFN-β (PBL Biomedical Laboratories, Piscataway, NJ).
  • ELISAs using commercial matched antibody pairs were obtained to further detect human IFN-α2b , CCL4 , CXCL8 and CXCL10 (B ).
  • CCL5 was detected by sandwich ELISA using a rabbit anti-human CCL5 antibody fromwith a goat anti-human secondary antibody from& .
  • ELISA plates were developed using theELISA amplification system according to manufacturer's instructions, and were acquired on a microplate reader and analysed using SoftMaxPro software .

Enzyme-Linked Immunosorbent Assay (ELISA)

  • An ELISA kit was used according to the manufacturer's protocol to detect IFN-β (PBL Biomedical Laboratories, Piscataway, NJ).
  • ELISAs using commercial matched antibody pairs were obtained to further detect human IFN-α2b , CCL4 , CXCL8 and CXCL10 (B ).
  • CCL5 was detected by sandwich ELISA using a rabbit anti-human CCL5 antibody fromwith a goat anti-human secondary antibody from& .
  • ELISA plates were developed using theELISA amplification system according to manufacturer's instructions, and were acquired on a microplate reader and analysed using SoftMaxPro software .

Gα12-dependent regulation of IL-6 and IL-8 in OSCC.

recombinant human IL-8
  • The secreted proteins of IL-6 and IL-8 are up-regulated by Gα12 in OSCC cells.

Chemotaxis assay

  • A migration assay was performed in 48- or 96-well Corning Costar transwell chambers with porous polycarbonate membranes with a pore size of 5 um .
  • hMSC at passage 4 were resuspended at 1 × 106/mL in the migration medium supplemented with 1% BSA and insulin transferrin selenium (ITS) and seeded in the upper chamber.
  • The following human recombinant proteins were used as chemoattractants in the lower compartment: hepatocyte growth factor (HGF), platelet-derived growth factor-AB (PDGF-AB), epidermal growth factor (EGF), VEGF-121, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF-1), macrophage inflammatory protein-3β (MIP-3β), macrophage inflammatory protein-1α (MIP-1α), B cell attracting chemokine-1 (BCA-1), regulation upon activation normal T cell express sequence (RANTES), growth regulated proteinα (GROα), fractalkine, SDF-1α, IL-1β, IL-6, IL-8, TNF-α .
  • The concentration of each cytokine was optimized to ensure maximal cell migration through the porous membrane.
  • The concentrations were: 40 ng/mL HGF, 10 ng/mL PDGF-AB, 10 ng/mL EGF, 10 ng/mL VEGF-121,…
  • A migration assay was performed in 48- or 96-well Corning Costar transwell chambers with porous polycarbonate membranes with a pore size of 5 um .
  • hMSC at passage 4 were resuspended at 1 × 106/mL in the migration medium supplemented with 1% BSA and insulin transferrin selenium (ITS) and seeded in the upper chamber.
  • The following human recombinant proteins were used as chemoattractants in the lower compartment: hepatocyte growth factor (HGF), platelet-derived growth factor-AB (PDGF-AB), epidermal growth factor (EGF), VEGF-121, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF-1), macrophage inflammatory protein-3β (MIP-3β), macrophage inflammatory protein-1α (MIP-1α), B cell attracting chemokine-1 (BCA-1), regulation upon activation normal T cell express sequence (RANTES), growth regulated proteinα (GROα), fractalkine, SDF-1α, IL-1β, IL-6, IL-8, TNF-α .
  • The concentration of each cytokine was optimized to ensure maximal cell migration through the porous membrane.
  • The concentrations were: 40 ng/mL HGF, 10 ng/mL PDGF-AB, 10 ng/mL EGF, 10 ng/mL VEGF-121, 10 ng/mL bFGF, 30 ng/mL IGF-1, 10 ng/mL MIP-3β, 50 ng/mL MIP-1α, 5 ng/mL BCA-1, 150 ng/mL RANTES, 50 ng/mL GROα, 300 ng/mL Fractalkine, 150 ng/mL SDF-1α, 10 ng/mL IL-1β, 100 ng/mL IL-6, 50 ng/mL IL-8 and 50 ng/mL TNF-α.
  • The chambers were incubated for four hours at 37°C in hypoxic or normoxic incubators.
  • The cells from the top side of the membrane chamber were removed and the migrated cells on the underside of the membrane were completely dislodged by incubating the membrane inserts in 15 mM ethylenediaminetetraacetic acid (EDTA) for 20 minutes.
  • All migratory cells were lysed with Triton X-100 and detected by CyQuant® GR dye according to a standard protocol .
  • Results were expressed as the mean number of net migrated cells over control cells (basal migration without chemotactic stimulus).
  • Each condition was tested in four wells; each experiment was repeated four times.

Read more

Isolated microparticles can be quantified and the real-time generation of neutrophil microparticles can be assessed with ISX.

IL-8
  • (a) Neutrophils were labeled with BODIPY-Maleimide, then microparticles were generated using TNF-α (50 ng/ml), IL-8 (50 ng/ml) or leukotriene B4 (10 nM) and enumerated using ISX.

Analysis of the functional activities of protein antigens expressed in the lumen of OMVs – (a) Haemolytic activity of Slo-OMVs.

human IL-8
  • 10 µg of SpyCEP-OMVs and “empty” OMVs were permeabilized with Triton X-100 and subsequently incubated with IL-8.

Cell Culture and Treatments

  • In experiments investigating CXC-chemokine signaling, cells were incubated in serum-free, phenol-red free RMPI 1640 or medium'>DMEM medium for 16h prior to exposure to 3nM recombinant human (rh)-CXCL8, 100ng/ml rh-CXCL12 or 100ng/ml rh-CCL2 .
  • For inhibition of chemokine signaling, cells were treated with RS102895 (CCR2 antagonist, 10nM, , ), AMD3100 (CXCR4 antagonist, 25μg/ml) or a CXCR1/2-derived pepducin (x1/2pal-i3, 300nM, , ).
  • Control cells were treated with the appropriate vehicle or NT-pepducin (x1/2pal-con) sequence as required.

Migration patterns of human neutrophils in response to fMLP, LTB4, C5a and IL-8 A) Percentage of human neutrophils loaded in the cell-loading channel, which migrate towards or away from fMLP, LTB4, C5a and IL-8 at different concentrations.

IL-8
  • Human neutrophils migrate towards and away from C5a and IL-8 in similar proportions.

HlgAB and HlgCB induce pro-inflammatory responses, and single components HlgA and HlgC are immunomodulatory molecules (a, b) Oxidative burst of human neutrophils induced by 1 µM fMLP after priming with 10 ng mL−1 TNFα, 1 nM C5a, 10 nM IL-8, and (a) 0.50 to 4.00 nM HlgCB or (b) 0.03 to 0.25 nM HlgAB.

IL-8
  • (d – f) Calcium mobilization with (d) 0.1 nM C5a, (e) 0.5 nM IL-8 in human neutrophils and (f) 1.0 nM MCP-1 in human monocytes inhibited by 94.0 nM HlgA or HlgC.