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.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 o C and using a high effector cell-target cell ratio (75 : 1). A good correlation with the 51Cr release assay was obtained.A potential application of the flow cytometric cytotoxicity assay using whole blood instead of isolated lymphocytes is presented.
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+Ii) and internal pH (pH,) 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 cyto-
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.
INTRODUCTIONA problem with the study of drug metabolism and toxicity in experimental animals is extrapolating the relevance of the results to man. It is unethical to determine the toxic properties of chemicals by dosing these to humans but cell cultures prepared from human tissues could circumvent this problem. Liver cell culture is a popular model system to study mechanisms of hepatotoxicity in experimental animals (for reviews see 1 and 2). However, a limitation of this model is the rapid loss of cytochrome P-450 during the first 24 hours of hepatocyte culture (3)(4)(5). This is an obstacle since cytochrome P-450 mediated metabolism frequently determines the toxicity and carc~nogenicity of many natural as well as synthetic chemicals (6).In rat hepatocyte culture the loss of cytochrome P-450 can be prevented by including metyrapone or 1,2-cyanophenylimidazole (7) in the culture medium. Little is known about the effect of the culture conditions on the concentration of cytochrome P-450 in human hepatocytes and where results have been reported these are only on a few donors (8-10). The present study reports our experience with hepatocyte cultures prepared from seven human individuals and therefore, to the best of our knowledge, represents the largest study to be reported, The results show that, in contrast to hepatocyte cultures prepared from experimental animals (3,4) the majority (i.e. five) of the cultures prepared from human liver do not lose cytochrome P-450 during the first 24 hours of culture. Furthermore, where the loss of cytochrome P-450 did occur in culture the addition of metyrapone or 1,2-cyanophenylimidazole to the culture medium, which prevents the loss of the cytochrome in rat hepatocyte culture (7), also prevents the loss of the cytochrome in cultured human hepatocytes. MATERIALS AND METHODSIsolation of hepatocytes. Liver samples were obtained from renal transplant donors who had met traumatic death and were maintained on life support systems until the kidneys were removed. Donors were not receiving any chronic drug treatment but were given such drugs as necessary while on life support systems. After removal of the kidneys a section of liver (approximately 100 g) was removed from the organ such that the section had only one cut face. In order to remove as much of the entrapped blood as possible, the liver section was perfused with Hank's balanced salt solution (HBSS from Gibco Europe) via the larger vessels on the cut surface, using a 20 ml syringe and a flow rate of ca 20 ml/min. The procedure to isolate hepatocytes commenced within 30 minutes of removal of the liver section from the donor. A smaller section (approx 30 g) was cut, ensuring that the section had only one cut face and this piece was used for the isolation of hepatocytes. A vessel was selected, so that a cannula with a male luer end-piece fitted snugly into it. Too large a vessel resulted in leakage of perfusate, too small a vessel resulted in the vessel being damaged and both resulted in poor perfusion. The liver section was perfuse...
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