Suspensions of Dictyostelium discoideum amoebae display free-running light scattering oscillations at the onset of development. We describe a device to monitor these oscillations in several samples in parallel. The apparatus consists of a thermostated cuvette holder where up to eight cuvettes containing cell suspension are inserted. Cells are aerated and kept in suspension via an airlift. Infrared light emitted from a five-diode array passes through the suspension and is detected by an array of five light detecting diodes. The resulting signal is digitized and recorded with a sampling rate of two measuring points/second. The parallel analysis approach allows determination of the effects of adding of agents or of variations in the external conditions in the same batch of amoebae at the same developmental time point. This represents an advantage over the conventional single cuvette approach, as oscillation characteristics themselves are developmentally regulated. Moreover, as the new experimental setup enables simultaneous analyses of up to eight samples, the behavior of wild-type and several mutant strains can be compared under identical experimental conditions.

Introduction

During differentiation single Dictyostelium discoideum amoebae aggregate and form a multicellular organism. The chemoattractant mediating aggregation and development, cAMP, is secreted periodically by cells in the center of the aggregate. Neighboring cells respond by an oriented, rhythmic inward movement and secrete cAMP themselves to relay the signal. In suspensions of amoebae, rhythmic changes in morphology result in altered light transmittance when analyzed photometrically. Spike-shaped and sinusoidal light scattering oscillations in cell suspensions (1) are caused by shape changes of amoebae but are also due to reversible aggregation of the cells (2). Light scattering oscillations are accompanied by oscillations of other parameters such as extracellular and intracellular concentrations of cAMP as well as extracellular levels of Ca²⁺, K⁺, H⁺, or other components (for a review see Reference (3). The interconnection of these parameters and their hierarchic order are not yet fully understood.

One aspect hampering the characterization of the oscillatory network and the regulation of the events underlying light scattering oscillation is the fact that the approach has relied on photometric detection of oscillations in one sample only (1). However, oscillation characteristics change in the course of differentiation, therefore the measurement of several samples (e.g., mutant and wild-type cell suspensions) at the same developmental time point would greatly improve oscillation analysis. Moreover, parallel analysis would greatly facilitate the study of agents that interfere with signal transduction, since effects can vary depending on when in the oscillation phase the agent is added. Here we describe a multichannel apparatus for parallel monitoring of light scattering in up to eight Dictyostelium cultures.

Materials and Methods

Cell Culture

D. discoideum wild-type strain Ax2 and the phospholipase C knockout mutant (PLC^-) (4) were grown as previously described (5), but without addition of vitamin B₁₂ and folic acid. PLC^- cells (kindly provided by P.J.M. Van Haastert, University of Groningen, The Netherlands) grew in the presence of 10 µg/mL G418. Use of Proteose Peptone No. 3 and of yeast extract (both manufactured by Difco, distributed by Becton Dickinson, Heidelberg, Germany) led to growth up to densities of 1.5–2 × 10⁷ cells/mL even in the absence of vitamins. Cells were washed by repeated centrifugation and resuspension of the cell pellet incold Sørensen phosphate buffer (13 mM KH₂PO₄/4 mM Na₂HPO₄, pH 6.0). Amoebae were shaken at 2 × 10⁷ cells/mL, 150 rpm, and 23°C until use.

Hardware

Schematics for the apparatus are shown in (Figure 1). The central components consist of eight aluminum blocks with milled depressions serving as cuvette holders that are mounted on a plastic base (dimensions 445 × 75 × 5 mm), the data acquisition unit, and the computer interface. The aluminum blocks contain the measuring units and are temperature controlled by input of water from a thermostated water bath. Aluminum or plastic plates can be inserted between the individual blocks, thus allowing thermal coupling or insulation of the cuvettes. Each cuvette is thermostated individually; four inflow and outflow tubes each are connected to the cuvette holders, and the diameter and length of the tubing is identical between the different holders in order to avoid temperature differences due to variations in tubing. Cuvettes were made from plexiglass and had an airlift system consisting of a stream of air emitted from a metal tube to simultaneously mix and aerate the cell suspension. Each cuvette was connected to the 0.3 bar compressed air supply by Teflon® tubing (0.2 mm inner diameter × 0.6 m). Since all other tubing leading to the air supply has a diameter at least 10-fold wider, the length and diameter of the Teflon tubing defines the total resistance to the upstream air pressure, and the effective aeration of each cuvette is therefore essentially independent of the number of neighbor cuvettes filled with cell suspension. Light scattering interference by the ascending air bubbles was prevented by a black plastic plate that partially separated the chamber into two compartments. Guides were milled in opposing sides of the cuvette, and the plate was positioned so that it did not interfere with light scattering readings. The base of the cuvette was rounded to ensure complete mixing of the cell suspension. The infrared signal from the array of Model HSDL-4420-1L1 light emitting diodes (Agilent Technologies, Palo Alto, CA, USA) was generated by an AC voltage with a duty cycle of 2 kHz. The current of the array of Model HSDL-5420-1L1 infrared light detecting photodiodes (Agilent Technologies) was adjusted potentiometrically to ensure similar illumination intensities in the individual cuvettes. The light detecting photodiodes detected light in synchrony with the duty cycle of the light emitting diodes. The difference in signal intensity between the on and off point of the duty cycle was preamplified (Model LF 356; produced by National Semiconductor) and rectified (demodulator Model AD 630IN, manufactured by Analog Devices; photodiodes, preamplifier and demodulator were bought from Conrad Electronics, Hirschau, Germany). This strategy renders the system insensitive to other, potentially interfering, light sources such as daylight. The preamplified analog signal was fed into a voltmeter and further amplified. Both offset and gain of the amplifiers were adjusted potentiometrically, which permitted preselection of the signal intensity range of interest. Subsequently, the signal (0–5 V bandwidth) was digitized either via a 12-bit A/D card (0–4096 steps; Conrad Electronics) for transfer by an RS232 (serial cable) interface to a computer or via an AD-USB-4 16-bit data acquisition system (Conrad Electronics) for data transfer via a USB port.