COX-2 ELISA Kit

The Calbiochem COX-2 ELISA Kit is intended for the quantitative measurement of COX-2 in human cell lysates. Serum and plasma samples are not recommended due to inherently low concentrations of COX-2 in biological fluids.

Cell lysates: Most mammalian cells do not express significant amounts of COX-2, so in order to detect COX-2 in lysates of most cell types; COX-2 must be induced by an appropriate stimulus. Below is a sample protocol for COX-2 induction in U937 cells:1. Prepare all reagents immediately prior to use. 2. Grow U937 cells in complete RPMI 1640 medium.3. Harvest the cells during the log phase of growth.4. Adjust the cell density to 1 x 10⁶ cells/ml and incubate for 3 days in the presence of 15 nM PMA.5. Harvest the cells and incubate for 24 h in fresh complete RPMI 1640 medium.6. Stimulate the cells with LPS for 6 h at a concentration of 250 ng/ml.7. Lyse the cells with CytoBuster Protein Extraction Reagent (Cat. No. 71009) according to the recommended protocol.8. Determine the protein concentration. A concentration of 2-5 mg/ml is recommended.9. Store the cell lysate at -70°C until use.

Note: Allow all kit components and samples to reach room temperature (18-25°C) prior to assay. Prepare all reagents immediately prior to use. The following table provides reagent preparation instructions to obtain a volume of reagent sufficient for one 2x8-well strip (16 wells).4. Standards:a. Reconstitute the COX-2 Standard with 1000 µl deionized or distilled water to yield a stock solution of 2000 ng/ml. Dispense unused reconstituted COX-2 Standard into aliquots and freeze (-70°C). Aliquots are stable for several months at -70°C.b. Prepare a standard curve by making dilutions of the reconstituted COX-2 Standard in Assay Diluent as follows:

Upon arrival store the entire contents of the kit at -20°C.

Positive Control
U937 cells

Anti-COX-2 Coated 96-Well Plate, COX-2 Standard, COX-2 Detector Antibody, HRP Conjugate, Assay Diluent, ELISA 20X Plate Wash Concentrate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol

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CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Toxicity: Multiple Toxicity Values, refer to MSDS (O)