Real time assays for quantifying cytotoxicity with single cell resolution.

A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly

DNA-coated AFM cantilevers for the investigation of cell adhesion and the patterning of live cells

Measurement of receptor adhesion strength requires the precise manipulation of single cells on a contact surface. To attach live cells to a moveable probe, DNA sequences complementary to strands displayed on the plasma membrane are introduced onto AFM cantilevers (see picture, bp=base pairs). The strength of the resulting linkages can be tuned by varying the length of DNA strands, allowing for controlled transport of the cells

Real Time Assays for Quantifying Cytotoxicity with Single Cell Resolution

<div><p>A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.</p></div

Programing cellular adhesion using DNA hybridization.

<p>(a) The exposure of live cells to an activated DNA reagent results in the rapid attachment of single strands to proteins in the cytoplasmic membrane. (b) Cell-substrate adhesion occurs within 5–20 min upon contact with a surface that displays the complementary DNA sequence. (c) The result of this process is shown for a human T-cell cancer line (Jurkat) bound to a 5 mm DNA spot printed on a glass slide. Unbound cells were readily washed from the slide. (d) When combined with DNA patterns generated using photolithography, ordered rectilinear arrays of cell clusters can also be produced. Both patterns are available in glass-bottomed chamber slides or 96-well plates.</p

Antibody-based cytotoxicity measured in whole blood.

<p>The scale bar in each image represents 100 µm. (a) Raji lymphoma cells were labeled with a blue Cell Tracker dye and then immobilized. Varying concentrations of α-CD20 were added, followed by freshly drawn human blood. The targeted cells could be easily identified using fluorescence microscopy, even in the presence of a large excess of blood cells. (b) After 16 h, the amount of cell death could be quantified using a PI stain (red). (c) A merged image allowed the ratio of living (blue) to dead (purple) targeted cells to be determined. (d) The level of whole blood cytotoxicity is shown for α-CD20, in addition to a control antibody (red point). The error bars indicate the standard deviation of two replicate experiments.</p

Heterotypic cellular microarrays for the determination of CDC specificity.

<p>Cell Tracker-stained Jurkat (blue channel, bottom left) and Jeko-1 (green channel, top right) cells were co-immobilized on cell microarrays. Following a CDC assay using 10 µg/mL α-CD20, the dead cells were stained red with PI (upper left). The Jeko-1 fluorescence overlapped significantly with the PI stain (yellow cells, bottom right), showing the degree of specificity of α-CD20 for CD20+ lymphoma cells. Overall, 76% of the Jeko-1 cells and 4% of the Jurkat cells were killed. The scale bar in each image represents 50 µm.</p

Cell-array CDC assays.

<p>(a) Microarrays of treated Jeko-1 cells were stained using a solution of propidium iodide (PI) after 4 h. The dead cells were imaged using a fluorescence microscope and counted using Image-J software, or quantified using an ImageXpress Micro automatic imager. The merged bright field and PI images facilitated the unambiguous identification of the dead cells (see close-up image in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066739#pone-0066739-g003" target="_blank">Figure 3a</a>). The images labeled “NC Ab” correspond to a control antibody that did not bind the targeted cells. The scale bar in each image represents 100 µm. (b) A dose-dependent response was observed for an anti-CD20 antibody (α-CD20). The error bars represent the standard deviation of three replicate experiments. (c) Using time-lapsed imaging, the number of dead cells can be determined as a function of time. In this representation, 100% represents the total number of dead cells observed at 4 h, which corresponds to 60% of the total cell population (as indicated on the plot in b). These data correspond to the video that is available as supporting information.</p

Images from a CDC assay using FITC-labeled α-CD20 at 10 µg/mL.

<p>Arrayed target cells (Jeko-1) were incubated with the antibody in human serum for 4 h, and then stained with PI. (a) A merge of the bright field and red-fluorescence (PI) images clearly indicated the dead cells. (b) The green fluorescence channel indicated that all of the target cells were bound by α-CD20. Comparison of the two images indicated which cells resisted the CDC mechanism. The scale bar in each image represents 50 µm.</p