Automatic Quantitation of cell Growth and Determination of Mitotic Index using Dapi Nuclear Staining

The growth rate of primary tumors and derived cell lines is an important identifying trademark to researchers studying the growth and differentiation of pediatric neoplasms. For the in vivo tumor specimen, the mitotic index is utilized to assess growth potential while, in vitro, the determination of the growth curve and doubling time of the cells is employed. Because of the importance of these parameters this laboratory has developed a technique for the simultaneous determination of cell number and mitotic index for adherent cultured cells, as well as solid tumors, using the polycationic dye DAPI (4′, 6-diamino-2-phenylindole). DAPI, when bound to nuclear DNA, fluoresces, and this property has been utilized to develop an image analysis based technique to determine cell number automatically based on gray scale discrimination. In addition, DAPI staining clearly delineates mitotic figures and this has allowed the simultaneous manual determination of mitotic index for each automatic cell count. This technique was first tested using four different human cell lines and was shown to determine cell growth and mitotic index simultaneously. Lastly, employing an atypical mesoblastic nephroma, it was shown that the mitotic index of the primary tumor could easily be obtained and compared to both the mitotic index of tumor heterotransplant in nude mice and the mitotic index of the tumor-derived cell culture. This technique should be useful in studies assessing the effects of various factors on the growth of adherent cultured cells as well as the accurate determination of the mitotic index in solid tumors.