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

A simple optical fiber device for quantitative fluorescence microscopy of single living cells

A simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultiplier are converted to voltage, amplified, and displayed on a recorder. In the excitation pathway a shutter control unit is mounted. With this control unit the period that the excitation pathway is `opened¿ and `closed¿ can be adjusted, to reduce cell damage and/or bleaching of the probe. This option allows time-lapse recording of experiments up to 1 h. We have used this set-up with a single and dual emission fluorescent probe to determine intracellular calcium concentrations and pH, respectively. In Fluo-3-loaded K562 target cells bound to natural killer cells, a temporary rise in [Ca2+]i was accompanied by bleb formation. The simple construction of this set-up is interchangeable between different types of fluorescence microscopes and can easily be combined with other microscopy techniques, e.g., patch clamp

Flow Cytometric Measurement of [Ca2+]i and pHi in Conjugated Natural Killer Cells and K562 Target Cells during the Cytotoxic Process1,2

We describe a flow cytometric assay that enables one to follow conjugate formation between cytotoxic cells and their target cells during the cytotoxic process. In addition, the internal calcium concentration ([Ca2+]i) and internal pH (pHi) of the conjugated cells can be monitored and directly compared to the nonconjugated cells. This is achieved by labeling one cell type with the Ca2+-specific dye Fluo-3, while the other cell type is labeled with the pH-sensitive dye SNARF-1. As these fluorochromes have different emission spectra, events positive for both fluorochromes are identified as conjugates. The results show that the conjugates can be clearly distinguished from single cytotoxic cells [natural killer (NK) cells] and target cells [K562 cells, (TC)]. Upon binding, [Ca2+]i is increased in the NK cells as well as in the TC. In conjugated NK cells this increase of [Ca2+]i is temperature dependent and is followed by a decrease to a normal [Ca2+]i value later on. The [Ca2+]i in NK cells increases in 2 steps, which may be related to the binding-and lethal hit phase. Upon conjugate formation, NK cells show a slight increase in pHi (0.2-0.3 pH units). TC do not reveal a significant change in pHi

A simple electronic device for time-lapse recording of neural and other cell movements using a home video cassette recorder

This article describes a simple electronic unit to obtain time-lapse recordings with the use of a common remote-controlled home video cassette recorder, for example a VHS recorder. The electronic unit is a timer to be connected to the remote-control unit. The video cassette recorder itself remains unchanged. Replay of the recorded images speeds up the original process by a factor of 2-100 × or more. This technique has been applied in video micrographic studies of (1) the development of dorsal root ganglion (DRG) cells in culture, including growth cone and Schwann cell movements, and (2) tumor cell killing by natural killer (NK) cells