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Cox2 (D5H5) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #13596
Gallery: Cox2 (D5H5) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #13596
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
Cox2 (D5H5) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) recognizes endogenous levels of total Cox2 protein.Species Reactivity: Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His108 of human Cox2 protein.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometry analysis in mouse cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated Cox2 (D5H5) XP® Rabbit mAb #12282.
Cyclooxygenase1 (Cox1) and cyclooxygenase2 (Cox2), family members with 60% homology in humans, catalyze prostaglandin production from arachidonic acid (1,2). While Cox1 expression is constitutive in most tissues, Cox2 expression is induced by lipopolysaccharide (LPS) and peptidoglycan (PGN) (3). PGN activates Ras, leading to phosphorylation of Raf at Ser338 and Erk1/2 at Tyr204. The activation of MAP kinase signaling results in subsequent activation of IKKα/β, phosphorylation of IκBα at Ser32/36, and NF-κB activation. Finally, activation of the transcription factor NF-κB is responsible for the induction of Cox2 expression (4). Investigators have shown that LPS and PGN induce the clinical manifestations of arthritis and bacterial infections, such as inflammation, fever, and septic shock (5). Research studies have indicated that Cox1 and Cox2 may also play a role in the neuropathology of Alzheimer's disease by potentiating γ-secretase activity and β-amyloid generation (6).
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. Alexa Fluor is a registered trademark of Life Technologies Corporation. The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS). This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is contingent on the buyer using the purchased product solely in research, including use with HCS or other automated imaging applications but excluding use in combination with DNA microarrays. The buyer must not sell or otherwise transfer this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; (c) manufacturing or quality assurance or quality control, or (d) resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or email@example.com.