Label ATTO-488. Maximum absorption 501 nm; maximum fluorescence 523 nm. The fluorescence is excited most efficiently in the 480 – 515 nm range. This label is analogous to the well known dye fluorescein isothiocyanate (FITC) and can be used with filters typically used to detect FITC.
Storage before reconstitution The antibody ships as a lyophilized powder at room temperature. Upon arrival, it should be stored at -20°C.
Reconstitution 50 µl double distilled water (DDW).
Antibody concentration after reconstitution 1 mg/ml.
Storage after reconstitution The reconstituted solution can be stored at 4°C, protected from the light, for up to 1 week. For longer periods, small aliquots should be stored at -20°C. Avoid multiple freezing and thawing. Centrifuge all antibody preparations before use (10000 × g 5 min).
Standard quality control of each lot Western blot analysis (unlabeled antibody, #ASR-013), and direct flow cytometry (labeled antibody).
Applications: FC
Direct flow cytometry
Human Jurkat living cells 5-10 µg/5x105 cells.
Cell surface detection of S1PR3 in human Jurkat intact live cells:
Sphingosine 1-phosphate (S1P) is an active byproduct of sphingomyelin metabolism. All cells can synthesize this biomolecule but the majority of its synthesis comes from erythrocytes and endothelial cells. In cases of inflammation, mast cells and platelets are the main source of sphingosine 1-phosphate1. S1P cellular functions could be intracellular, where it is synthesized. In addition, upon secretion, it circulates in the blood via its binding to high-density lipoproteins and albumin. When S1P reaches its target cell it could activate 5 different high affinity receptors belonging to the G-protein coupled receptor superfamily: Sphingosine 1-phosphate receptors, termed S1PR1-51.
Stimulation of S1P receptors triggers a cascade of signaling events depending on the receptor and on the G-protein it couples. S1PR1 couples to Gi2. S1PR2 can couple to Gs, Gq or G12/13 and S1PR3-5 can couple to Gior G12/133-4. The pathways activated vary from Ca2+ mobilization, activation or inhibition of adenylate cyclase phospholipase C activation and more. Through the different signaling pathways these receptors activate, S1P1 receptors are implicated in adherens junction assembly, cytoskeletal changes, cell migration, proliferation and apoptosis5.
S1PR3 is specifically detected in the brain, heart, spleen, liver, lung, thymus, kidney, testis and skeletal muscle3. S1PR1 and S1PR2 are generally expressed in the CNS, cardiovascular and immune systems1. S1PR4 is specifically expressed in the lymphoid tissue and S1PR5 in natural killer cells and olygodendrocytes1.
Malfunction of S1P receptor signaling is reported in various disorders, for example multiple sclerosis and may be targets for the development of therapeutic drugs1.
Application key:
CBE- Cell-based ELISA, FC- Flow cytometry, ICC- Immunocytochemistry, IE- Indirect ELISA, IF- Immunofluorescence, IFC- Indirect flow cytometry, IHC- Immunohistochemistry, IP- Immunoprecipitation, LCI- Live cell imaging, N- Neutralization, WB- Western blot