Cluster analysis of flow cytometric list mode data on a personal computer

A cluster analysis algorithm, dedicated to analysis of flow cytometric data is described. The algorithm is written in Pascal and implemented on an MS-DOS personal computer. It uses k-means, initialized with a large number of seed points, followed by a modified nearest neighbor technique to reduce the large number of subclusters. Thus we combine the advantage of the k-means (speed) with that of the nearest neighbor technique (accuracy). In order to achieve a rapid analysis, no complex data transformations such as principal components analysis were used. \ud Results of the cluster analysis on both real and artificial flow cytometric data are presented and discussed. The results show that it is possible to get very good cluster analysis partitions, which compare favorably with manually gated analysis in both time and in reliability, using a personal computer

A new principle of cell sorting by using selective electroporation in a modified flow cytometer

When a strong electric field pulse of a few microseconds is applied to biological cells, small pores are formed in the cell membranes; this process is called electroporation. At high field strengths and/or long pulse durations the membranes will be damaged permanently. This eventually leads to cell kill. \ud We have developed a modified flow cytometer in which one can electroporate individual cells selected by optical analysis. The first experiments with this flow cytometer were designed to use it as a damaging sorter; we used electric pulses of 10 s and resulting field strengths of 2.0 and 3.2 X 106 V/m to kill K562 cells and lymphocytes respectively. The hydrodynamically focused cells are first optically analyzed in the usual way in a square flow channel. At the end of this channel the cells are forced to flow through a small Coulter orifice, into a wider region. If optical analysis indicates that a cell is unwanted, the cell is killed by applying a strong electric field across the Coulter orifice. The wanted living cells can be subsequently separated from the dead cells and cell fragments by a method suitable for the particular application (e.g., centrifugation, cell growth, density gradient, etc.). \ud The results of these first experiments demonstrate that by using very simple equipment, sorting by selective killing with electric fields is possible at rates of 1,000 cells/s with a purity of the sorted fraction of 99.9%

A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells

A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate, F-18) prior to incubation with the effector cells. F-18 remains in the membrane of the target cells even when they are killed thereby permitting a clear separation between effector and target cells. Dead cells are determined by staining with a second fluorochrome (propidium iodide) after incubation of effector and target cells.\ud F-18 is not toxic and does not decrease the cytotoxic activity of human natural killer cells. It is also stable (exchange between labeled and non-labeled cells is negligible in a period of at least 4 h at 37°C) and it remains in the membrane of the killed cells. A clear distinction between unlabeled effector and labeled target cells is obtained, even after incubation of target and effector cells for 4 h at 37°C and using a high effector cell-target cell ratio (75:1). A good correlation with the 51Cr release assay was obtained.\ud A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.\ud \u

A flow cytometric study of the membrane potential of natural killer and k562 cells during the cytotoxic process

This study demonstrates that it is possible to investigate the membrane potential of interacting cells during the cytotoxic process using flow cytometry. Changes in the membrane potential of NK and K562 cells, involved in a cell-mediated cytotoxic process, were studied by standard and slit-scan flow cytometry, using the membrane potential sensitive fluorescent probe DiBAC4(3). The NK cells were labeled with a membrane marker (TR-18 or DiI) prior to incubation with K562 cells and the conjugates that were formed could be identified on the basis of the membrane marker fluorescence and light scattering signals. With a slit-scan technique we measured the membrane potential of each cell in a conjugate separately. The results show that depolarization of the K562 cell occurs as a consequence of the cytotoxic activity of the NK cell. This depolarization appears to be an early sign of cell damage because the cell membrane still remains impermeable to propidium iodide. Our data also indicate that depolarization of the NK cell occurs as a result of its cytotoxic activity

Wigner crystals in two-dimensional transition-metal dichalcogenides: Spin physics and readout

Wigner crystals are prime candidates for the realization of regular electron lattices under minimal requirements on external control and electronics. However, several technical challenges have prevented their detailed experimental investigation and applications to date. We propose an implementation of two-dimensional electron lattices for quantum simulation of Ising spin systems based on self-assembled Wigner crystals in transition-metal dichalcogenides. We show that these semiconductors allow for minimally invasive all-optical detection schemes of charge ordering and total spin. For incident light with optimally chosen beam parameters and polarization, we predict a strong dependence of the transmitted and reflected signals on the underlying lattice periodicity, thus revealing the charge order inherent in Wigner crystals. At the same time, the selection rules in transition-metal dichalcogenides provide direct access to the spin degree of freedom via Faraday rotation measurements.Comment: 15 pages, 12 figure