Purinergic Signalling (2008) 4:357–364 The information on the gradient shape based on the carboxyfluorescein measurements is a reasonable estimate for the gradients in the growth-cone-turning experiments, as diffusion coefficients of carboxyfluorescein and the inves- tigated compounds (mainly adenosine) are comparable. This is suggested by two lines of evidence. The diffusion coefficient can be calculated on the basis of the Stokes- Einstein radius [29], which is related to molecular weight and molecular volume. Both values are in the same range for adenosine (molecular weight: 267.24; molecular vol- ume: 128.1 cm3/mol) and carboxyfluoresceine (molecular weight: 332.3; molecular volume: 207.4 cm3/mol) [30]. Further evidence comes from literature: while Rani et al. [31] give a diffusion coefficient for fluoresceine of 4.9×10–6 cm2 s-1 (21.5°C in water), Mohrman [32] concludes that the diffusion coefficient of adenosine is similar to that of sucrose, which is 5.1×10–6 cm2 s-1. Optical setup An inverted epifluorescence microscope (IX70-S1F, Olym- pus, Tokyo, Japan) equipped with a 40× oil immersion objective (UAPO 40× OL/340/1.35, Olympus, Tokyo, Japan) was used throughout the study except for the TIRF measure- ments. The setup was equipped with a CCD camera (IMAGO- VGA) for time-lapse microscopy and was controlled by TILLvisIon (version 4.0), which was also used for image analysis. All imaging equipment was supplied by TILL Photonics (Gräfelfing, Germany) unless otherwise noted. Measurement of extension and turning of growth cones Neurite growth was analysed according to Ming et al. [33] including the modifications of Munck et al. [14]. In short, the growth direction before the onset of the stimulus was aligned with the y-axis of a coordinate system. Turning angles were determined by measuring the angles between the ordinate and a straight line connecting the origin of the coordinate system (starting point of the growth cone) with the final growth-cone position. Positive angles were defined to be on the right side of the ordinate (stimulated side) and negative angles to be on the left side. Data were statistically analysed using the Mann-Whitney U test. Deviations are given as standard error of the mean (SEM). All growth- cone-turning experiments lasted 15 min. Neurites that retracted during this period were not included in further analysis. Chemicals The following chemicals were used in growth-cone-turning assays: ADO, NECA, CGS 21680, CGS 15943, R-(-)-PIA, KT 5720 (Calbiochem-Novabiochem, San Diego, CA, 359 USA), and Rp-cAMPS (Biomol, Hamburg, Germany). Stock solutions of these chemicals were prepared in dimethyl sulfoxide (DMSO), with the exception of the water soluble Rp-cAMPS. If not stated otherwise, chem- icals were bought from Sigma (St. Louis, MO, USA). Results Cultures of DRG sensory neurons from embryonic chickens have been used by various groups to investigate different aspects of growth-cone turning [14, 22–25, 34, 35]. Herein explant cultures of chicken (E 11) DRGs were used to investigate a possible effect of ADO on the direction of growth-cone advance. During time-lapse image recordings of elongating neurites, the growth cones were exposed to extracellular gradients of ADO and related compounds. The effect of these concentration gradients on growth direction was analysed on the basis of growth tracks that were extracted by image analysis from the images of the time- lapse recordings (cf. “Materials and methods”). Generation and characterisation of micro-gradients Constraints for effective guidance with soluble compounds are that the concentration falls between certain maximum and minimum limits, and that the percentage change in concentration across the width of the growth cone exceeds a certain minimum value [36]. Therefore, we characterised the pipettes and the respective gradients produced with it. Here extracellular concentration gradients were generated by gravity-driven flow of the respective compound (or vehicle control) from a micro-pipette. Two pipette types were compared, as the gradient shape and gradient evolution depend on the geometry of the pipette. The inner tip diameter was analysed with raster electron microscopy and for the small pipette type was typically 1 µm and for the larger type 3 µm (Fig. 1). The gradients generated with these pipettes were investigated by TIRF-microscopic measurements as described in “Materials and methods”. These experiments show that the gradients stabilise within 5 min and stay stable for the duration of the experiments, and that the gradient properties depend on pipette geometry. Using the small pipette filled with a 1 mM solution of carboxyfluorescein, the dye concentration decreased from about 1 μM at a distance of 5 μm to 0.6 μM at a point 50 μm distant from the pipette. The corresponding data for the large pipette were 17 μM at 5 μm to 1.5 μM at 50 μm (Fig. 1). Similar diffusion coefficients of carboxyfluores- cein and ADO allowed the estimation of the ADO concentrations at different distances from the pipette tip on the basis of the carboxyfluorescein data. These estima- tions resulted in ADO concentrations of 100 nM at a