Vala Sciences

MDCK cells were cultured in 96-well dishes under the indicated conditions then visualized for pan-cadherin using Vala’s kit reagents; the cells were then photographed using a high content microscopy workstation (Beckman IC100 Image Cytometer) using a 20X objective, with 4 images acquired per well. A, Control cells visualized for cadherins (green) and for nuclei (DAPI, blue) utilizing Vala’s pan-cadherin reagent kit. B, Image mask representing the plasma membrane derived from the pan-cadherin image by Vala’s CyteSeer® software. C, cells exposed to EGTA (3 mM) for 3 hrs, visualized for pan-cadherin and nuclei as in A. D, Mean total intensity of pixels in the pan-cadherin representing the membrane mask (TMM). TMM was quantified for every cell in each image using Vala’s CyteSeer software; the values were then averaged across all cells in each well and further averaged across all wells per condition. E, The ratio of the median pixel intensity of pan-cadherin at the membrane mask (MMM) to the median pixel intensity of the cytoplasm (MMC). F, The Mander’s K2 split overlap coefficient (Manders et al., 1992) for the pan-cadherin and DAPI. For D – F, each bar represents the mean ± standard deviation for 6 wells; **, p < 0.01; Z’ = 1-3(SDCon+SDEGTA)/absolute(MeanCon-MeanEGTA) (Zhang et al., 1999) .

    To illustrate the functionality of Vala’s pan-cadherin reagents and our Windows, Mac, and Linux-compatible, CyteSeer® image cytometry software, MDCK cells were plated to confluence on 96-well dishes, then exposed to culture media ± 3 mM EGTA for 3 hrs. The cells were then visualized using our pan-cadherin kit reagents and imaged using a digital microscopy workstation (Figure 1). For cells cultured in control medium, cadherins are localized primarily at junctions between neighboring cells (Figure 1A), where they function to provide cell-to-cell adhesion and linkage to the cytoskeleton. For cells visualized for pan-cadherin and nuclei using Vala’s kit reagents, CyteSeer® did an excellent job of segmenting the images, assigning pixels to the plasma membrane (Figure 1B), as well as the nuclear and cytoplasmic compartments (data not shown). Since cell adhesion is a calcium-dependent interaction between extracellular domains of cadherins on neighboring cells, chelating extracellular calcium with EGTA leads to dissociation of the cells from each other, and redistribution of cadherins (Figure 1C).

    For images of cells labeled for membrane proteins such as cadherins, and for nuclei using stains specific for DNA (e.g., DAPI) CyteSeer®’s Membrane analysis algorithm automatically derives 88 separate data parameters related to cell size and shape, intensity of the pixels in the membrane, cytoplasmic, and nuclear compartments, and colocalization between the labels in each of these compartments. The data are derived on a cell-by-cell basis and can be conveniently averaged across multiple images obtained for each well in a high content screening experiment. For the experiment with EGTA, the total integrated intensity of pixels at the plasma membrane (TMM) was significantly diminished by the experimental treatment (Figure 1D). Additionally, the ratio of median intensity at the membrane to the median intensity within the cytoplasmic compartment (R:MMM/MMC) was also diminished (Figure 1E) and this data was extremely consistent for each condition, yielding a positive Z’ value for the comparison between control and EGTA-treated cells (see Figure 1 legend for derivation of Z’). Finally, the Mander’s K2 split overlap coefficient (one of several colocalization coefficients calculated on a cell by cell basis by CyteSeer®), calculated between the pan-cadherin image and the DAPI image, was also increased by EGTA in a very consistent manner (Figure 1F), confirming EGTA-induced redistribution of pan-cadherin and increased colocalization between the pan-cadherin and DAPI images. The high Z’ value obtained for the Mander’s K2 coefficient (Z’ = 0.65) indicates that the assay is exceptionally robust and suitable for high throughput screens in which hundreds of thousands of compounds could be tested for effects on cadherin expression and distribution. Thus, the Vala Sciences Inc Pan-cadherin reagent kit, along with Vala’s CyteSeer® software can be used to quantify changes in overall expression levels and cellular localization of cadherins, a critically important family of adhesion proteins.

HeLa cells cultured in 96-well dishes, were incubated with various concentrations of PMA, an agent that elicits translocation of PKC from the cytoplasm to the plasma membrane then visualized for nuclei (blue), pan-cadherin (red), and PKC (green) using Vala Sciences reagents. Control cells are shown in panels A-C; cells exposed to 100 nM PMA are shown in panels D-F. The Pearson’s correlation coefficient, calculated between the PKC and pan-cadherin images by Vala’s CyteSeer® software is shown in G. Symbols and error bars represent the mean ± standard deviation for 8 wells per condition.

    The pan-cadherin reagents can also be used in multi-plexed assays with other Vala reagents, with the goal of quantifying the colocalization of various proteins with cadherins at the plasma membrane. For example, Protein Kinase Cα, a member of the conventional PKC family, is found in the cytoplasm of unstimulated cells, and migrates to the plasma membrane in response to hormones that elicit increases in intracellular calcium and increased diacylglycerol formation, as commonly occurs with hormone binding to G-protein coupled receptors. Phorbol 12-myristate 13-acetate (PMA) is a diacylglycerol mimetic, which, when added to cells, causes translocation of PKC to the plasma membrane. To quantify this translocation, HeLa cells were incubated with various concentrations of PMA for 15 minutes, then visualized for both cadherins and PKC; Vala’s CyteSeer® software was then used to quantify the colocalization of PKC with the pan-cadherin image. Since cadherins remain at the plasma membrane during the exposure to PMA, increased translocation of PKC to the plasma membrane results in increased colocalization of PKC with the cadherins. Indeed, the Pearson’s coefficient of correlation, calculated between the PKC and pan-cadherin images, displayed an excellent dose-dependent increase in response to PMA, with a Z’ value of 0.56 (Figure 2G).

A, Skin tissue section visualized for nuclei (blue), pan-cadherin (green), and keratin 14 (red). The centrally located keratin-14 expressing cells are a tumor. B, A longer exposure of the same field of view of A is shown for the pan-cadherin channel (note diminished expression of cadherins in the tumor). C, Mask derived for the pan-cadherin image in A by CyteSeer® . D, Quantification of pan-caderin expression in keratin negative (K14-) vs. keratin positive (K14+). Bars represents the mean ±SE for n= 48 or 83 cells, respectively. For details see Prigozhina et al., 2007.

    Vala’s pan-cadherin reagents can also be utilized to visualize cadherins in tissue slices. Cadherins are often downregulated in tumor cells. To analyze this relationship, a tissue section obtained from a murine inducible skin tumor model was colabeled for pan-cadherins and for keratin 14 (Prigozhina et al., 2007). Analysis of the resulting image by CyteSeer® enabled a cell-by-cell analysis of cadherin expression as a function of keratin 14 expression, which serves as a tumor marker in this system (Figure 3).

    Vala Science’s pan-cadherin kits are available in different versions to accommodate multi-plexing with other assays based on either mouse monoclonal, or rabbit polyclonal antibodies. Kits can also be requested that visualize pan-cadherin in either the red or green fluorescence channel.
    The goal of Vala Sciences Inc is to develop products enabling quantitative image cytometry for academic and pharmaceutical researchers. For further information please contact Vala via our webpage, email, or phone.

Geiger, B., Volberg, T., Ginsberg, D., Bitzur, S., Sabanay, I. 1990. Broad spectrum pan-cadherin antibodies, reactive with the C-terminal 24 amino acid residues of N-cadherin. J. Cell Science 97:607-614.

Manders, E. M., Verbeek, F. J., Afen, J. A. 1993. Measurement of colocalisation of objects in dual colour confocal microscopy. J. Microscopy 169:375-382

Morishita, H., Yagi, T. 2007. Protocadherin family: diversity, structure, and function. Current Opinion in Cell Biology 19:584-592.

Prigozhina, N. L., Zhong, L., Hunter, E. A., Mikic, I., Callaway, S., Roop, D. R., Mancini, M. A., Zacharias, D. A., Price, J. H., and

McDonough, P. M. 2007. Plasma membrane assays and three-compartment image cytometry for high content screening. ASSAY and Drug Development Technologies 5: 29-48.

Zhang et al., 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomolecular Scr. 4:67-73

Vala Sciences is a cell biology company offering software, kits, services and custom development for analyzing a wide variety of cell types and conditions from adipocytes & stem cells to primary & well established cell lines.

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