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

SUMMARY: To evaluate the effect of several cytokines on the total production of insoluble ECM, we performed immunoperoxidase staining directly on rat mesangial cells cultured on flat-bottomed 96-well plates. the peroxidase activity was demonstrated by orthophenilenediamine (OPD) and measured directly as optic density at 490 nm (OD490) by a microplate reader. After this procedure, cell 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 OD490 was corrected by OD540 (OD490/OD540). OD540 was linearly correlated with actual cell number in the well and OD490 for each ECM was firmly correlated 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 recombinant interferon gamma (IFNγ). Human recombinant platelet derived growth 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 evaluation for other proteins of insoluble form as well as ECM produced by attached form of cultured cells.