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13797S 100 µl (50 tests) $329.00.0
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REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of Raw 264.7 cells, untreated (blue) or LPS-treated (100 ng/ml, 6 hr; green), using IL-6 (D5W4V) XP® Rabbit mAb (Mouse Specific) (PE Conjugate). All cells were incubated with Brefeldin A #9972 (300 ng/ml, last 3 hr of treatment).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibodies. Protect from light. Do not freeze.

Specificity / Sensitivity

IL-6 (D5W4V) XP® Rabbit mAb (Mouse Specific) (PE Conjugate) recognizes endogenous levels of total IL-6 protein.


Species Reactivity: Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant mouse IL-6 protein.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in mouse cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated IL-6 (D5W4V) XP® Rabbit mAb (Mouse Specific) #12912.


Acute phase response is induced by interleukin-6 (IL-6) produced by T cells, macrophages, fibroblasts, endothelial and other cells (1,2). IL-6 induces proliferation or differentiation in many cell types including B cells, thymocytes and T cells. IL-6, in concert with TGF-β, is important for developing Th17 responses. IL-6 binds to IL-6Rα and through this association induces gp130 homodimerization (1). gp130 homodimerization triggers the Jak/Stat cascade and the SHP-2/Erk MAP kinase cascade (1,3,4). IL-6 also forms a complex with an IL-6Rα splice variant that is nonmembrane-associated (3). The IL-6/soluble IL-6Rα complex can then activate the gp130 signaling pathway in cells that express gp130 but not IL-6Rα (3). Research studies have shown that IL-6, through increasing expression of proangiogenic VEGF, may also contribute to metastatic breast cancer (5).


1.  Heinrich, P.C. et al. (1998) Biochem J 334 ( Pt 2), 297-314.

2.  Heinrich, P.C. et al. (1998) Z Ernahrungswiss 37 Suppl 1, 43-9.

3.  Jones, S.A. (2005) J Immunol 175, 3463-8.

4.  Jenkins, B.J. et al. (2004) Mol Cell Biol 24, 1453-63.

5.  Hong, D.S. et al. (2007) Cancer 110, 1911-28.


Entrez-Gene Id 16193
Swiss-Prot Acc. P08505


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

13797
IL-6 (D5W4V) XP® Rabbit mAb (Mouse Specific) (PE Conjugate)