Axio observer z

ROS levels were quantitatively assessed using dihydroethidine (DHE) and MitoSOX™-Red, and mitochondria were co-stained with MitoTracker^® Green FM (all purchased from Molecular Probes, Eugene, OR, USA). Cells were washed with endothelial basal medium and incubated with either 10 µM DHE or 5 µM MitoSOX™-Red combined with 100 nM MitoTracker^® Green FM for 30 min at 37 °C. Afterward, cells were washed twice with endothelial basal medium, and images were taken with an Axio Observer Z (Zeiss, Jena, Germany) using a 200× magnification. Fluorescence intensity was calculated with Image J and normalized to the cell number.

Immunostaining of ECs was performed as previously described [12 (link)]. In detail, cells were fixed with 4% formaldehyde for 15 min and were blocked and permeabilized for 15 min at room temperature with 3% (v/v) normal goat serum (Sigma-Aldrich, Deisenhofen, Germany) diluted in PBS containing 0.3% (v/v) Triton X-100. Afterward, the cells were incubated with primary antibodies against a myc-tag or p21 (all from Cell Signaling Technologies, Frankfurt, Germany), each 1:100, overnight at 4 °C. The antibody against TIM23 (BD BioSciences, Heidelberg, Germany) was diluted 1:150 and incubated overnight at 4 °C. Subsequently, cells were washed three times with PBS and incubated with an Alexa Fluor^® 594 coupled anti-mouse or anti-rabbit secondary antibody (1:500) (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h at room temperature. Nuclei were counterstained with DAPI (500 ng/mL) (Carl Roth, Karlsruhe, Germany) in PBS for 5 min at room temperature and the cells were mounted with ProLong™ Diamond Antifade Mountant (Invitrogen, Darmstadt, Germany). Images were taken using Zeiss microscopes (Axio Observer Z or Axio Imager M2, Zeiss, Jena, Germany) using a 400× or 200× magnification. Pixel intensities were measured with Image J [17 (link)].

Cell cluster areas were quantified as described in reference 18 (link). Briefly, samples were imaged in 96-well glass-bottomed plates (Ibidi 89621) at ×10 magnification using transmitted light (bright field) on a Zeiss Axio Observer.Z1/7 widefield microscope with a Hamamatsu Orca-Flash 4.0 LT CMOS digital camera. Images from 3 biological replicates were processed and analyzed with the following functions in ImageJ: “smooth” to reduce bacterial background, “find edges” to further highlight choanoflagellate cells, “make binary” to convert to black and white, “close-” to fill in small holes, and “analyze particles” to calculate the area of each cell cluster. Particles smaller than 10 μm² were removed to reduce background bacterial signal. P values were calculated using a nonparametric Kolmogorov-Smirnov test in Prism software (GraphPad).