Visualization and quantification of cytotoxicity mediated by antibodies using imaging flow cytometry.

Conventional approaches for the detection of antibody dependent cell-mediated cytotoxicity (ADCC) activity rely on quantification of the release of traceable compounds from target cells or flow cytometry analysis of population-wide phenomena. We report a new method for the direct imaging and quantification of ADCC of cancer cells. The proposed method using imaging flow cytometry combines the statistical power of flow cytometry with the analytical advantages of cell imaging, providing a novel and more comprehensive perspective of effector/target cell interactions during ADCC events. With this method we can quantify and show in detail the morphological changes in target and effector cells, their apoptotic index, the physical interaction between effector and target cells, and a directional transfer of cytosolic contents from effector to target cells. As a model system we used the therapeutic anti-CD20 antibody rituximab to target CFSE labeled Ramos human Burkitt's lymphoma cells, to CMTPX-labeled human monocytic U-937 effector cells. We expect that similar studies using different effector and target cell populations may contribute to the pre-clinical evaluation of therapeutic antibodies and help to identify mechanisms that could be beneficial in the immunotherapy of cancer.

Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods.

Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation. Here a novel method is presented for accurate quantitation of apoptosis based on nuclear condensation, nuclear fragmentation, and membrane blebbing using automated image analysis on large numbers of images collected in flow by the ImageStream multispectral imaging cytometer. Additionally the measurement of nuclear fragmentation correlates with the secondary methods of detection of apoptosis over time, indicating that it is also an early marker for apoptosis. False-positive and false-negative events associated with each photometric flow cytometry based method are quantitated and can be automatically removed/included where appropriate. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms allowing more complete and robust analysis of apoptosis.

Biochemical characterization of MmoS, a sensor protein involved in copper-dependent regulation of soluble methane monooxygenase.

Methane monooxygenase (MMO) enzymes catalyze the oxidation of methane to methanol in methanotrophic bacteria. Several strains of methanotrophs, including Methylococcus capsulatus (Bath), express a membrane-bound or particulate MMO (pMMO) at high copper-to-biomass ratios and a soluble MMO (sMMO) form when copper is limited. The mechanism of this "copper switch" is not understood. The mmoS gene, located downstream of the sMMO operon, encodes a sensor protein that is part of a two-component signaling system and has been proposed to play a role in the copper switch. MmoS from M. capsulatus (Bath) has been cloned, expressed, and purified. The purified protein is a tetramer of molecular mass 480 kDa. Optical spectra indicate that MmoS contains a flavin cofactor, identified as flavin adenine dinucleotide (FAD) by fluorescence spectroscopy and chromatographic analysis. The redox potential of the MmoS-bound FAD, which binds within the N-terminal PAS-PAC domains, is -290 +/- 2 mV at pH 8.0 and 25 degrees C. Despite extensive efforts, MmoS could not be loaded with Cu(I) or Cu(II), indicating that MmoS does not sense copper directly. These data suggest that MmoS functions as a redox sensor and provide new insight into the copper-mediated regulation of sMMO expression.