Analysis of Cell Ploidy Utilizing High-Content Screening Techniques
Cell populations that are actively dividing contain cells with 2X or even greater multiples of the normal diploid DNA content. Analysis of DNA content is very actively pursued in biomedical research applications involving cancer and genomics research (Ross et al., 2003; Harada et al., 2005). Vala Sciences CyteSeer® image processing platform software, which is compatible with virtually any computer and images acquired with any digital microscope workstation, has recently been enabled to perform ploidy analysis for cultured cells stained with fluorescent indicators that bind to DNA (e.g., DAPI).
To illustrate the capability of CyteSeer to analyze ploidy, human dermal microvascular endothelial cells (HDMECs), plated in a 96-well glass-bottomed dish were cultured in the presence of 10% fetal bovine serum (FBS) or serum-started overnight. The cells were then stained for DNA with DAPI and imaged on a Beckman IC100 high content screening robotic microscopy workstation. Since the amount of DAPI that binds to each cellular nuclei is proportional to DNA content, analysis of the Total Nuclear Intensity (TNI) of each cell provided an index of ploidy. For each condition, 8 wells were analyzed with 4 images taken per well. HDMECs cultured under standard, growth promoting conditions (10 % FBS) had a prominent peak most likely representing cells with a 2N content (typical diploid cells) for the bins between 150000 to 180000 TNI (Figure 1). Additionally, there was a second peak, for bins between 280,000 and 360,000 that likely represents cells with a DNA content of 4N (11.9% of the total population). For cells cultured overnight in low serum, the population of 4N cells was reduced to 6.3% of the total population. These data are consistent with the known effect of serum-withdrawal to reduce cell division for cultured primary cells.
Figure 1. Ploidy analysis of human dermal microvascular endothelial cells utilizing CyteSeer.
Data represent analysis of total nuclear intensity in the DAPI channel for 738 cells cultured under control conditions (10% FBS) vs 568 cells cultured overnight in low serum (2% FBS).
These data illustrate that Vala Sciences’ CyteSeer software can be utilized for ploidy analysis of cultured cells. Vala’s goal is to develop CyteSeer into an affordable, general purpose image cytometry program, that will be useful for Academic and Pharmaceutical researchers working in Cell and Molecular Biology. For further information, please visit our website at Valasciences.com, phone at 858 581 6275, or email to info@valasciences.com.
References
Harada, J., N., Bower, K. E., Orth, A. P., Callaway, S., Nelson, C. G., Laris, C., Hogenesch, J., Vogt, P. K., Chanda, S. K. 2005. Identification of novel mammalian growth regulatory factors by genome-scale quantitative image analysis. Genome Res. 15:1136-1144.
Ross, J. S., Linette, G. P., Stee, J., Ross, M. S., Anwar, S., Boguniewicz, A. 2003. DNA ploidy and cell cycle analysis in breast cancer. Am. J. Clin. Pathol. 120 Suppl: S72-S84.
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