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H Endogenous Rabbit IgG

Flow Cytometry

Flow cytometric analysis of THP-1 cells, undifferentiated (blue) or differentiated (green) by treatment with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (80 nM) overnight and Human Interleukin-4 (hIL-4) #8919 (100 ng/ml) for 48 hours after TPA, using DC-SIGN (D7F5C) XP® Rabbit mAb (PE Conjugate) (solid lines) and a concentration matched isotype control Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).

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Flow Cytometry

A. Solutions and Reagents

  1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
  2. Formaldehyde (methanol free).
  3. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100mL 1X PBS. Store at 4°C.

B. Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
  3. Fix for 10 minutes at 37°C.
  4. Add 5 ml PBS and rinse by centrifugation.
  5. Resuspend cells in 5 ml PBS.
  6. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
  3. Resuspend cells in 100 µl incubation buffer per assay tube.
  4. Block in Incubation Buffer for 10 min at room temperature.
  5. Add fluorochrome-conjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody datasheet or product webpage for the appropriate dilution).
  6. Incubate for 1 hr at room temperature.
  7. Wash by centrifugation in 2–3 ml incubation buffer.
  8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

posted January 2009

Protocol Id: 180

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

DC-SIGN (D7F5C) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total DC-SIGN protein. This antibody does not cross-react with DC-SIGNR.

Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human DC-SIGN protein.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated DC-SIGN (D7F5C) XP® Rabbit mAb #13193.

DC-SIGN (CD209, CLEC4L) is a C-type lectin receptor expressed by dendritic cells (DCs) (1,2). The DC-SIGN transcript can undergo several splicing events to generate at least thirteen different transmembrane and soluble isoforms (3). DC-SIGN responds to a broad range of pathogens due to its ability to recognize both mannose and fructose carbohydrates, and is well studied for its role in HIV infection. Recognition of the HIV envelope glycoprotein gp120 by DC-SIGN leads to internalization of HIV by DCs and facilitates transmission of the virus to CD4+ T cells (2,4). DC-SIGN also mediates adhesion to T cells through interaction with ICAM-3, as well as transmigration across the endothelium by binding to ICAM-2 (1,5). The DC-SIGN receptor can modulate TLR signaling by activating the kinase Raf-1 (6,7). The closely related molecule DC-SIGNR (L-SIGN, CLEC4M) is 77% homologous to DC-SIGN and likely arose through a gene duplication event (8). Like DC-SIGN, DC-SIGNR binds mannose carbohydrates on the surface of pathogens (8,9). However, the expression patterns of the two receptors differ, as DC-SIGNR expression is restricted to endothelial cells of the liver, lymph node, and placenta (10). Murine cells contain a set of related molecules, SIGNR1-SIGNR8 (11). Based on sequence analysis, there is no clear murine ortholog to human DC-SIGN, however SIGNR3 is the most functionally similar due to its ability to recognize both mannose and fructose structures (11).

1.  Geijtenbeek, T.B. et al. (2000) Cell 100, 575-85.

2.  Geijtenbeek, T.B. et al. (2000) Cell 100, 587-97.

3.  Mummidi, S. et al. (2001) J Biol Chem 276, 33196-212.

4.  Kwon, D.S. et al. (2002) Immunity 16, 135-44.

5.  Geijtenbeek, T.B. et al. (2000) Nat Immunol 1, 353-7.

6.  Gringhuis, S.I. et al. (2007) Immunity 26, 605-16.

7.  Gringhuis, S.I. et al. (2010) Nat Immunol 11, 419-26.

8.  Bashirova, A.A. et al. (2001) J Exp Med 193, 671-8.

9.  Mitchell, D.A. et al. (2001) J Biol Chem 276, 28939-45.

10.  Pöhlmann, S. et al. (2001) Proc Natl Acad Sci U S A 98, 2670-5.

11.  Powlesland, A.S. et al. (2006) J Biol Chem 281, 20440-9.

Entrez-Gene Id 30835
Swiss-Prot Acc. Q9NNX6

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.

DC-SIGN (D7F5C) XP® Rabbit mAb (PE Conjugate)