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CD44 (156-3C11) Mouse mAb (PE Conjugate) #8724
Gallery: CD44 (156-3C11) Mouse mAb (PE Conjugate) #8724
Flow Cytometry, Methanol Permeabilization 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.
- 16% Formaldehyde (methanol free).
- 100% methanol.
- Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
- Fix for 15 min at room temperature.
- Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.
- Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
- Incubate 30 min on ice.
- Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.
- Aliquot desired number of cells into tubes or wells.
- Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
- Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
- Incubate for 1 hr at room temperature. Protect from light.
- 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 optional DNA stain (Section E).
E. 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 July 2009
revised June 2017
CD44 (156-3C11) Mouse mAb detects endogenous levels of total CD44 protein.Species Reactivity: Human
Monoclonal antibody is produced by immunizing BALB/c mice with stimulated human leukocytes and recognizes residues surrounding Ser210 of human CD44
This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated CD44 (156-3C11) Mouse mAb #3570.
CD44 is a type I transmembrane glycoprotein that mediates cell-cell and cell-matrix interaction through its affinity for hyaluronic acid (HA) and possibly through other parts of the extracellular matrix (ECM). CD44 is highly polymorphic, possesses a number of alternative splice variants and undergoes extensive post-translational modifications (1,2). Increased surface levels of CD44 are characteristic of T cell activation, and expression of the protein is upregulated during the inflammatory response. Research studies have shown that interactions between CD44 and HER2 are linked to an increase in ovarian carcinoma cell growth (1-3). CD44 interacts with ezrin, radixin and moesin (ERM), linking the actin cytoskeleton to the plasma membrane and the ECM (4-6). CD44 is constitutively phosphorylated at Ser325 in resting cells. Activation of PKC results in phosphorylation of Ser291, dephosphorylation of Ser325, disassociation of ezrin from CD44, and directional motility (4).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.