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

Abstract

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.

14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

“…The number of mitotic cells were quantified based the method as described [66]. Briefly, 96 well collagen coated plates were used to seed cells at a final concentration of 1000 cells/well in their respective media.…”

“…Cells were then incubated for three days at 37°C in 3% or 21% O 2 . Finally, cells were washed, resuspended in phosphate buffered saline and stained with DAPI, as described [66]. Images of stained cells were acquired using a Zeiss Axiovert 200M inverted fluorescent microscope using 10X magnification and Openlab (PerkinElmer) image acquisition software.…”

“…Images of stained cells were acquired using a Zeiss Axiovert 200M inverted fluorescent microscope using 10X magnification and Openlab (PerkinElmer) image acquisition software. Using Image J, a set threshold for staining intensity was used to count the brightly stained nuclei, with obvious chromatin condensation and the mitotic index was determined based on the ratio of number of mitotic cells present in 1000 cells, as described [66].…”

Statistical methods for estimating doubling time in in vitro cell growth

“…Because of its simplicity, doubling time has been widely used to compare the growth pattern of cells. In vitro doubling time of the cells is calculated for differentiation in cell lines derived from pediatric neoplasms (18) and for comparing the different characteristics of clones (3). Doubling time is also used to discriminate the cell growth pattern in breast cancer cells (2,8,9,13), adult human brain cells (4), endometrial tissue (6), smooth muscle cells (12,17), pancreatic carcinoma cells (14), and hmnan tracheal gland ceils (16).…”

Acceleration of Myofiber Formation in Culture by a Digitized Synaptic Signal

“…Phasecontrast and corresponding fluorescence images were captured at 10 · magnification under identical conditions. To facilitate quantification of healthy versus pyknotic nuclei, [22][23][24][25][26] reactive pixels were quantified using ImageJ with minimum arbitrary thresholds set first at 50 arbitrary densitometric units (to visualize all nuclei) followed by 100 (which restricts visualization to denser, pyknotic nuclei).…”