Heme Oxygenase 1 Activation Kit

The kit contains a mouse monoclonal heme oxygenase 1 primary antibody, a DyLight™ 549-Conjugated Secondary Antibody, and various other reagents and buffers required for immunofluorescent detection for high-content screening (HCS) assays.

Heme oxygenase 1 is a microsomal enzyme that catalyses the oxidation of heme to antioxidants, biliveridin and carbon monoxide and protects cells from wide variety of stress conditions through activation of p38 MAPK. Carbon monoxide produced during oxidation of heme by heme oxygenase 1 activates p38 MAPK, which confers tissue protection through inhibition of cytokine production. Heme oxygenase 1 can be induced by oxidative stress, hypoxia, heat shock, heavy metals and cytokines.

To measure heme oxygenase 1 levels, A549 cells were treated with copper sulfate. In normal cells, heme oxygenase 1 protein levels are low. Upon activation, heme oxygenase 1 increases in the cytoplasm and nucleus, as evidenced by increased staining. The assay was developed in A549 cells and optimized using the Thermo Scientific ArrayScan HCS Reader and Compartmental Analysis Bioapplication Software Module. Stained cells also can be imaged using fluorescence or confocal microscopy.

Figure 2. Staining of Heme oxygenase 1 in A549 cells treated with or without 0.5 mM copper sulfate for 24 hours. Cells were stained according to the kit protocol and imaged using the ArrayScan® HCS Reader.

Figure 3. Dose response curve of heme oxygenase 1 in A549 cells treated with copper sulfate for 24 hours. Data represents mean ± SD from three plates (eight wells per 96-well plate per dose of copper sulfate).