Fluoview fv1000 confocal laser

To determine whether PPI affects HaCaT cell proliferation, an EdU incorporation assay was carried out. 48 hours after cell seeding, 10 μM EdU was poured into the culture following incubation for some time followed by fixation using 4% paraformaldehyde in PBS for 15 minutes. EdU labeling with an azide derivative of Apollo 643 was performed using a Cell-Light™ EdU Apollo 643 In Vitro Imaging Kit (^#C10310-2, RiboBio Co., Ltd., China). A 652 nm laser was used for the excitation of Apollo 643. Microscopic images were obtained with a FluoView FV1000 confocal laser scanning microscope (Olympus, Japan). ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to get composite images.

Confocal images with a resolution of 1024 × 1024 pixels were acquired on a FluoView FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan), equipped with a 60× UPLSAPO 1.35 NA oil immersion objective (Olympus), in sequential scanning mode, and with a 2× or 3× zoom depending on the experiment carried out. The pixel dwell time was 12.5 μs and images were processed with three times Kalman line averaging. All images were saved in Olympus Original Imaging Format (OIF), which includes greyscale TIF file data. Unbiased, automatic quantifications of images for analysis of organelle number and intensity were performed with CellProfiler 2.1.1 software [24 (link)] pipelines, in which individual cells were segmented to perform per cell measurements.

Embryogenic callus were induced from inflorescences of selected three PvSPL6-GFP_OE lines. These callus was cultured on SM5 medium {MS0 + 5 mg/L 2,4-D (2,4-Dichlorophenoxyacetic acid) + 0.15 mg/L 6-BA [N-(Phenylmethyl)-9H-purin-6-amine]} supplemented with different hormones and plant growth regulators for 2 weeks. For hormones and growth regulators treatments, different concentrations of 2,4-D (0, 1, 3, and 5 mg/L), 6-BA (0.02, 0.05, 0.1, and 1 mg/L), 6-Furfurylamino-purine (KT; 0, 0.5, 1, and 4 mg/L), gibberellin (GA₃; 0, 10, 100, and 400 mg/L), and paclobutrazol (0, 0.5, 1, and 2 mg/L) were used, respectively. The fluorescence signal of each callus type under each hormone and growth regulator treatment was observed 48–72 h later using a FluoView FV1000 confocal laser scanning microscope (Olympus, Japan).