Dp controller 1 2 1108

Embryogenesis of Astrephomene gubernaculifera 2014-1002-YkAs8 and Eudorina sp. 2010-623-F1-E8 was observed by time-lapse light microscopy based on a method reported previously [9 (link)] with some modifications (Additional file 5: Figure S3). To examine the embryos from anterior–lateral, lateral, posterior–lateral and posterior angles, fully mature vegetative colonies of Astrephomene were fragmented into several parts using a Dounce tissue grinder (Wheaton Industries Inc., Millville, NJ, USA) and attached to coverslips coated with polyethylenimine. Then, the coverslips were placed on slides and sealed with Vaseline. Preparations were observed using a BX-53 microscope (Olympus, Tokyo, Japan) equipped with Nomarski interference optics. Plural photomicrographs with different optical sections were obtained using DP Controller 1. 2. 1108 (Olympus) at 1-min intervals with manual successive changes in focus. In Eudorina, as fragmentation of the colonies was not possible, fully mature colonies were directly attached to coverslips and observed as described above. Only anterior–lateral view images were obtained from Eudorina. The images of both species were analyzed and processed using ImageJ 1.50b (National Institutes of Health, Bethesda, MD, USA).

To estimate relative genome size of V. africanus and V. reticuliferus, 4’,6-diamidine-2-phenylindole (DAPI)-staining was performed using somatic cells of V. africanus, male and female strains of V. reticuliferus (2013-0703-VO2 and 2013-0703-VO3, respectively), and V. carteri strain EVE (control). One ml of each vegetative sample was fixed with 0.25% glutaraldehyde, followed by postfixation in 100% methanol for reducing autofluorescence, and washed with phosphate-buffered saline. Fixed samples were stained with 0.1μg/μl DAPI overnight. DAPI-stained somatic cells of V. africanus and V. reticuliferus male and female were mixed separately with DAPI-stained V. carteri strain EVE and mounted in the same slide. The images were obtained using a BX-60 Microscope and DP Controller 1. 2. 1108 (Olympus, Tokyo, Japan). The image analyses were performed using ImageJ, measuring the mean gray value of 10 nuclei for each exposure time (0.50, 0.67, 1.0, 1.5, 2.0 and 2.5 s).

[Ca²⁺] in the ER or cytosol was determined using G-CEPIA1er or GCaMP6f. Then 28 hr after transfection, Ca²⁺ imaging was performed with a fluorescence stereomicroscope (Olympus IX-71-22TFL/PH) and acquisition software (DP Controller 1.2.1.108). Fluorescence intensities were measured using ImageJ (https://imagej.nih.gov/ij/). After extracting the green channel, subtracting the background (rolling ball radius: 50.0 pixels), and applying threshold, average gray value of whole cells in each image was determined. Bradykinin, 4CmC, and CDN1163 were obtained from Abcam, Tokyo Chemical Industry, and Sigma-Aldrich, respectively.