Semiquantitative measurement of total extracellular matrix protein produced by cultured mesangial cells

To evaluate the effect of several cytokines on the total production of inso luble ECM, we performed immunoperoxidase staining directly on rat mesangial cells cultured on flat-bottomed 96-well plates. The peroxidase activity wa s demonstrated by orthophenilenediamine (OPD) and measured directly as opti c density at 490 nm (OD490) by a microplate reader. After this procedure, c ell number in each well was determined by crystal violet staining of which intensity was measured at 540 nm (OD540). The amount of ECM measured as OD4 90 was corrected by OD540 (OD490/OD540). OD540 was linearly correlated with actual cell number in the well and OD490 for each ECM was firmly correlate d with cell number in the well. By this method, dose dependent decrease of fibronectin (FN) and laminin (LM) was observed in the presence of rat recom binant interferon gamma (IFN gamma). Human recombinant platelet derived gro wth factor (PDGF) increased the total production of LM and type IV collagen (Col IV) as well as cell number. This method would also be useful in evalu ation for other proteins of insoluble form as well as ECM produced by attac hed form of cultured cells.