STAT6, phosphorylated (Y641) (APC) (STAT6B, STAT6C, D12S1644, IL-4-STAT, interleukin 4 induced STAT, Signal Transducer and Activator of Transcription 6)

Signal Transducer and Activator of Transcription 6 (STAT6) mediates the signaling of cytokines such as IL-4 and IL-13. STAT6 acts as a signal transducer in the cytoplasm and, upon phosphorylation at Y641, translocates to the nucleus and binds to the DNA consensus site TTCN4GAA. Knockout studies in mice suggest that STAT6 functions in differentiation of T helper 2 (Th2) cells, expression of cell surface markers, and class switch of immunoglobulins.\n\nIntended Use:\nThe reagent is designed for flow cytometric applications intended to identify and quantitate cells expressing cytoplasmic forms of the protein.\n\nPrinciple of the Test:\nFixed cells are permeabilized, allowing conjugated antibodies access to proteins within the cell. Cells are initially subjected to a fixation step in order to minimize leakage of proteins out of the cell. The conjugated antibody is allowed to penetrate and bind to its target within the cell. Following a final wash, cells are analyzed on a flow cytometer. Flow cytometric analysis of APC conjugates will generate a signal which can be detected using 620-650nm wavelength laser excitation and monitoring emitted fluorescence with a detector optimized to collect peak emissions at 660-670nm.\n\nSample Preparation:\nIntracellular staining antibodies are designed for multiparameter flow cytometric analysis of cells. To stain for surface proteins (e.g. CD3, CD4, CD8) in addition to the intracellular protein, we recommend that the investigator determine whether the fixation and permeabilization steps adversely affect the surface protein. If so, surface staining of cells prior to fixation and permeabilization is recommended.\n\nAdditional Reagents Required:\nParaformaldehyde Fixative-Dissolve 4.0 g of paraformaldehyde in 100 ml of sterile PBS (10 mM phosphate buffered saline, pH 7.4) by heating the solution at 56 degrees C for about 1 hour. All solids must be fully dissolved prior to use. Store buffer at 2 degrees -8 degrees C, protected from light, for no longer than 2 weeks.\n\nSample Staining:\n1. Harvest cells, and wash twice in PBS by spinning at 200 x g for 7 minutes.\n2. If cells are to be surface stained, follow the staining procedure indicated by the antibody manufacturer.\n3. Resuspend a maximum of 5 x 10e5 washed (or surface stained) cells in 0.5 ml of cold 4% paraformaldehyde fixative and incubate at room temperature for 10 minutes.\n4. Vortex the cells intermittently in order to maintain a single cell suspension. Following fixation, wash the cells twice in an isotonic PBS buffer supplemented with 0.5% BSA by centrifuging at 200 x g for 7 minutes.\n5. While vortexing, resuspend cells in 900 il ice-cold (-20 degrees C) methanol and incubate cells for 30 minutes at 4 degrees C.\n6. Centrifuge the cells at 200 x g for 7 minutes.\n7. Decant the supernatant. Wash twice in PBS/BSA buffer. After the second wash ensure that approximately 200il of buffer remains in the tube.\n8. Gently resuspend the cells in the remaining PBS/BSA buffer and add 10il (or a previously titrated amount) of antibody conjugate.\n9. Briefly vortex the tube and incubate for 30-45 minutes at room temperature in the dark.\n10. Wash the cells twice using 2 ml of PBS/BSA buffer each time. Centrifuge as in step 6.\nPhospho-STAT6 (Y641)-APCCell Count\n11. Resuspend the cells in each tube with 200-400il of PBS for final flow cytometric analysis.\nIntracellular staining of unstimulated cells (open histogram-dotted line) or rhIL-4-stimulated DAUDI cells (filled histogram) with APC-conjugated anti-Phospho-STAT6 (Y641) or with isotype control antibody.\n\nMay be stored at 4 degrees C before opening. DO NOT FREEZE! Stable at 4 degrees C as an undiluted liquid. Dilute only prior to immediate use. Stable for 12 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. Freezing APC conjugates will result in a substantial loss of activity. APC conjugates are sensitive to light.

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