Tom C. Bakker Schut

Intracellular carotenoid levels measured by Raman microspectroscopy: comparison of lymphocytes from lung cancer patients and healthy individuals

Most studies concerning a possible protective role of carotenoids against cancer focus on serum carotenoid levels. We have used Raman microspectroscopy to study the intracellular amounts of carotenoids in lymphocytes of lung cancer patients and of healthy individuals. Our results indicate a significant decrease of carotenoids in lung carcinoma patients compared with healthy individuals, particularly in adenocarcinoma patients. Carotenoid supplementation raised the serum concentration in 2 lung cancer patients up to normal levels, whereas intracellular content remained significantly lower. This indicates that carotenoid uptake by lymphocytes is not only dependent on serum carotenoid concentration. Our findings indicate that Raman microspectroscopy, a recently developed technique to measure intracellular levels of drugs, is also well suited to obtain quantitative data on carotenoid amounts inside cells. Int. J. Cancer 74:20–25.

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.

Flow cytometry signal processing and data acquisition with a personal computer using an RTI‐800 multifunction A/D I/O board

The design and implementation of a data management system, consisting of a real‐time data processing and a PC‐based acquisition system, for measurement of flow cytometric data is described. The data processing system makes use of an erasable programmable logic device for implementation of dedicated control logic. The signal processing system is used for measuring the peak heights of 8 analog signals and simultaneous digital measurement of the time of flight. Four window discriminators are used for data selection and sorting applications. The data acquisition system is based on an MS‐DOS PC with a slightly modified commerical A/D I/O board, which we used to measure 8 analog signals (12 bit A/D converted) and 32 bit digital information per event. Using an IBM compatible personal computer with an 8 MHz AT bus, the total system is capable of measuring about 10u2009000 events per second.

Manipulation and characterization of cells by laser light

Both for medical and biotechnological purposes it is very important to be able to analyze and manipulate cellular populations on a single cell basis. Moreover it is often important to be able to sort a subfraction of cells from the total population with defined specific characteristics. Since the retainment of biological viability is of importance in such cases the use of monoclonal antibodies for cell characterization cannot always be applied. The use of light scattering offers an attractive alternative. One of the manipulations which is at the beginning of many biotechnological processes is the fusion between two well characterized and differing cell types. In the present paper we concentrate on the use of flowcytometry for light scattering characterization and fusion of cells.