IL-6 Receptor antibody (PE-Cy7)

Catalog No. ABIN2659082

Product Details anti-IL-6 Receptor Antibody (PE-Cy7)

Target: IL-6 Receptor (IL6R) (Interleukin 6 Receptor (IL6R))

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This IL-6 Receptor antibody is conjugated to PE-Cy7

Application: Flow Cytometry (FACS)

Purification: The antibody was purified by affinity chromatography and conjugated with PE/Cy7 under optimal conditions. The solution is free of unconjugated PE/Cy7 and unconjugated antibody.

Clone: UV4

Isotype: IgG1 kappa

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Restrictions: For Research Use only

Independent Validation (FACS)

Validation #101960 (Flow Cytometry)

by: Immunmodulatorische Abteilung, Universitätsklinikum Erlangen

No.: #101960

Date: 11/06/2017

Antigen: IL6R

Lot Number: B236171

Method validated: Flow Cytometry

Positive Control: Mature dendritic cells (mDCs) from three donors, incubated with ABIN2659082

Negative Control: Mature dendritic cells (mDCs) from three donors, incubated without antibody

Notes:

Passed. ABIN2659082 specifically recognizes the IL-6 Receptor on mDCs.

Validation Images

A Flow cytometry analysis of mature DCs from three different donors after incubation for 30min or 120min with or without ABIN2659082. B Consecutive gating for single cells (left panel), mDCs (middle panel), and living cells (right panel), illustrated on donor 2 samples stained for 30min with ABIN2659082.

Full Methods

Primary Antibody: ABIN2659082

Full Protocol:

• Prepare peripheral blood mononuclear cells (PBMCs) from leukoreduction system chambers (LRSCs) (Pfeiffer et al. (2013); Weidinger et al. 2011) from three healthy donors (obtained following informed consent and approved by the institutional review board) by density centrifugation on a Lymphoprep gradient (Axis-shield AS, 1114547, lot 12IFS10).
• For the generation of dendritic cells (DCs), prepare PBMCs from leukoreduction system chambers (LRSC) (Pfeiffer et al. (2013)).
• After centrifugation, collect PBMCs forming a distinct band at the sample/Lymphoprep interface.
• Wash PBMCs 3x with cold PBS supplemented with 1mM EDTA.
• Wash PBMCs 1x with cold RPMI 1640 (Lonza, BE12-167F, lot 7MB088).
• Resuspend PBMCs in warm DC Medium (RPMI 1640 supplemented with 1% AB-serum, 100U/ml penicillin, 100mg/ml streptomycin, 2mM L-glutamine, 10mM HEPES buffer (Lonza, BE17-737E)) and seeded into 175cm² tissue culture flasks at a density of 3.5x10⁸ cells/flask.
• Let monocytes adhere for 1h at 37°C and 5% CO₂.
• Wash non-adherent fraction off with warm RPMI 1640.
• Add 30ml DC medium supplemented with 800U/ml recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and 250U/ml recombinant human interleukin-4 (IL-4) to each flask (day 1).
• Incubate cells for 72h at 37°C and 5% CO₂.
• On day 4 (or three days later), add 5ml of fresh culture medium containing GM-CSF (final concentration of 400 U/ml in 35ml) and IL-4 (final concentration of 250U/ml in 35ml) to the cells.
• On day 5, mature the immature DCs by adding 10ng/ml recombinant human tumor necrosis factor alpha (TNF-α), 1μg/ml prostaglandin E2 (PGE2), 200U/ml recombinant human interleukin-1β (IL-1β), 1000U/ml recombinant human interleukin-6 (IL-6), 40U/ml GM-CSF, and 250 U/ml IL-4 to the medium.
• Incubate cells for 48h at 37°C and 5% CO₂.
• On day 7, use the now mature DCs (mDCs) for FACS analysis.
• Analyze cell surface expression of IL-6 Receptor by FACS:
• • Harvest 2.5x10⁵ mDCs by centrifugation.
  • Wash cells once with FACS buffer (PBS supplemented with 2% FCS).
  • Combine anti-human PE-Cy7 conjugated IL-6 Receptor antibody (antibodies-online, ABIN2659082, lot B236171) diluted 1:100 and LIVE/DEAD Violet dead cell stain kit (ThermoFisher Scientific, L34955, lot 1832693) diluted 1:300 in cold FACS buffer.
  • Resuspend mDCs in 100µl of the antibody-solution and incubate for 30min on ice in the dark.
  • Wash mDCs 2x with cold FACS buffer.
  • Fix mDCs using 2% paraformaldehyde in cold PBS.
  • Store mDCs at 4°C until analysis.
  • Run samples using a BD FACS Canto II and analyze events using FCS Express 5.

Experimental Notes:

Mature DCs were stained for FACS for the analysis of the expression of IL-6 Receptor/CD126 on the cell surface of the cells with ABIN2659082 for 30min or 120min respectively. The histogram shows a clearly shift between unstained control and the stained mock control by staining for 120min. The staining for 30min does also show a shift of the histogram peak, but not as clear as the staining for 120min.

Handling

Buffer: Phosphate-buffered solution, pH 7.2, containing 0.09 % sodium azide and 0.2 % (w/v) BSA .

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: Protect from prolonged exposure to light. Do not freeze.

Storage: 4 °C

Storage Comment: The antibody solution should be stored undiluted between 2°C and 8°C.

Target Details for IL-6 Receptor

Target: IL-6 Receptor (IL6R) (Interleukin 6 Receptor (IL6R))

Alternative Name: CD126 (IL6R Products)

Synonyms: IL6R antibody, il-6ra antibody, CD126 antibody, IL-6R-1 antibody, IL-6RA antibody, IL6Q antibody, IL6RA antibody, IL6RQ antibody, gp80 antibody, IL6R1 antibody, Il6ra antibody, IL-6Ralpha antibody, IL-6R antibody, Il6r antibody, interleukin 6 receptor antibody, interleukin 6 receptor, alpha antibody, IL6R antibody, il6r antibody, Il6r antibody, Il6ra antibody

Background: CD126 is an 80 kD IL-6 receptor α chain also known as IL-6R. It is a member of the immunoglobulin superfamily that is expressed on plasma cells, T cells, activated B cells, monocytes, granulocytes, hepatocytes, epithelial cells, and fibroblasts. Functional IL-6 receptors are formed by the non-covalent association of CD126 and the IL-6 receptor β chain (CD130 or gp130). CD126 binds IL-6 with low affinity but does not signal. The β chain (gp130, CD130) does not bind IL-6 by itself but associates with the α-chain/IL-6 complex to initiate signal transduction. IL-6 binding to the receptor complex results in the stimulation of B and T cells, and hematopoietic precursor proliferation and differentiation. A soluble form of CD126 has been found in human serum.

Pathways: JAK-STAT Signaling, Autophagy, Growth Factor Binding, Cancer Immune Checkpoints