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DC-SIGN (D7F5C) XP® Rabbit mAb (PE Conjugate) #71481
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).Learn more about how we get our images
Gallery: DC-SIGN (D7F5C) XP® Rabbit mAb (PE Conjugate) #71481
Flow Cytometry, Extracellular Epitope Protocol for Direct Conjugates
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
- Formaldehyde (methanol free).
- Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) (#9998) in 100mL 1X PBS. Store at 4°C.
NOTE: If live cell staining is desired, proceed to Section C.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml PBS. Add formaldehyde to obtain a final concentration of 4% formaldehyde.
- Fix for 15 minutes at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 1X PBS.
- Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.
NOTE: Count cells using a hemocytometer or alternative method.
- Aliquot desired number of cells into tubes or wells.
- If necessary, centrifuge to remove excess PBS.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 30-60 minutes at room temperature.
- Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
- Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to Section D.
D. Optional DNA Dye
- Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
- Incubate for at least 5 min at room temperature.
- Analyze cells in DNA staining solution on flow cytometer.
posted January 2009
revised June 2017
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
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human DC-SIGN protein.
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).
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.