Quantification of Choanoflagellate Cell Clusters

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).