Cooled ccd camera

In order to visualize cell spreading on a substrate, 1.5x10⁵ cells were allowed to adhere for 20 min on a glass surface in a FluoroDish (World Precision Instr., Sarasota, FL). To monitor the presence and spreading of D. discoideum cells we used an inverted microscope (Olympus IX71 or Zeiss Axiovert 100M) and imaged by phase contrast and Reflection Interference Contrast Microscopy (RICM) as previously described [12 (link)]. Images and movies (15 frames per second) were acquired with an Olympus DP30 CCD camera or a High resolution black/white CCD camera (Hamamatsu CCD cooled camera). RICM images were sub-sampled at 1 image per 1.2 s, the background was subtracted and flattened and the noise filtered. Dark cell-surface contact zones were defined by segmentation and quantified as described [13 (link)].
To measure cell motility, cells were observed for 60 min (picture every 30 sec) by phase contrast with a Plan-Neofluar 10x magnification. Pictures were taken with a Hamamatsu CCD cooled camera. We used Particle tracking from the Metamorph software to track individual cell trajectories.

Annexin V binding to phosphatidylserine can be used as a marker of early apoptotic events^{60 (link)}. Binding of Cy5-conjugated annexin V to HCASMC was assessed using a kit (Biotium, USA). Cells were co-stained with Hoechst 33342 (Sigma, UK) to identify nuclei and visualised using a fluorescence microscope (Nikon, Japan) and images acquired using a cooled CCD camera (Hamamatsu, Japan). Equivalent numbers of cells were captured for each field. Fluorescence intensity was determined using analysis software (Image J, NIH, USA).

For Fura-2 Ca²⁺ imaging experiments, the cells were first loaded with 2 μM Fura-2-AM in presence of 0.02% of Pluronic-127 (both from Life Technologies) and 10 μM sulfinpyrazone (Sigma) for 20 min at RT. Fura-2 loaded cells were washed in KRB + Mg + Ca and allowed to de-esterify for 20 min before cLTP induction.
After that, the coverslips were mounted into acquisition chamber and placed on the stage of a Leica DMI6000 epifluorescence microscope equipped with S Fluor ×40/1.3 objective. The probe was excited by alternate 340 and 380 nm using a Polychrome IV monochromator and the Fura-2 emission light was filtered through 520/20 bandpass filter and collected by a cooled CCD camera (Hamamatsu, Japan). The fluorescence signals were acquired and processed using MetaFluor software (Molecular Device, Sunnyvale, CA, USA). To quantify the differences in the amplitudes of Ca²⁺ transients the ratio values were normalized using the formula ΔF/F0 (referred to as normalized Fura-2 ratio, “Norm. Fura ratio”). At least two coverslips for each of three independent culture preparation were imaged for each condition. In mixed neuron-astroglial cultures, astrocytes were recognized as flat polygonal or star-like cells, while neurons were recognized by round bodies with few processes located on upper focal plane above the astrocytes. The cells with uncertain morphology were not taken in consideration.