Axiovert 100 inverted microscope

Two strains from Bahía de La Paz (BAPAZ-7 and BAPAZ-5) and two strains from Bahía de Mazatlán (BAMAZ-2 and GCMQ-4) were used in this study (Figure 6). Table 3 shows the isolation information for each strain. Strain GCMQ-4 (Bahía de Mazatlán, Sinaloa) was acquired from the marine dinoflagellate collection (CODIMAR) of CIBNOR in La Paz, Mexico. The isolation information of the strains in this collection can be found at https://www.cibnor.gob.mx/investigacion/colecciones-biologicas/codimar accessed on 13 July 2022.
Vegetative cells were collected by vertical and superficial trawls with a plankton net (20 μm mesh). The concentrated phytoplankton collected in each trawl was passed through a 60 μm mesh net to remove larger organisms. The filtrate was inoculated in 250 mL containers with f/2 culture medium [106 (link)] that had been modified by adding selenium (10⁻⁸ M H₂SeO₃) and reducing the copper concentration (10⁻⁸ M CuSO₄) [107 (link)]. In the laboratory, vegetative cells of G. catenatum (Supplementary Material Figure S2) were isolated by single cell isolation with a capillary tube under an Axiovert 100 inverted microscope (Carl Zeiss, Oberkochen, Germany) and individually transferred to a 24-cell plate containing the modified f/2 medium. Cells were incubated at a temperature of 21.0 ± 1.0 °C, light intensity of 120 μmol photon m⁻² s⁻¹, and a 12:12 h light–dark cycle.

CHO-K1 cells stably expressing receptors of interest were transiently transfected with β-arrestin2-GFP using Lipofectamine 2000 (Invitrogen). Twenty-four hours post-transfection, cells were plated on 35-mm glass bottom plates. On the following day, cells were starved for at least 2 h in serum-free medium prior to stimulation. After stimulation, cells were fixed with 5% formaldehyde, and diluted in phosphate-buffered saline containing calcium and magnesium before analysis by confocal microscopy using an Axiovert 100 inverted microscope equipped with a C-Apochromat 63X/1.2 oil immersion objective (Zeiss). The 488-nm excitation beam of an Argon-Krypton laser and a 500–550-nm band-pass emission filter were used. The beam power was kept below 10% of maximal power to reduce photobleaching and phototoxicity.

MCF7 cells (2.5 × 10⁵) were seeded on 22 mm-Aclar plastic coverslips (Pro-Plastics Inc., Linden, NJ, USA) previously coated with rat-tail collagen. After treatment with NETs (500 ng/mL) for 16 h, cells were fixed with 4% paraformaldehyde diluted in PBS (pH 7.4), permeabilized with PBS containing 0.5% Triton X-100 and incubated with primary antibodies against: β-catenin (1:50, #C-2206, Sigma Chemical Co, Saint Louis, MO, USA), E-cadherin (1:50, #04-1103, Millipore, Burlington, MA, USA), fibronectin (1:50, #F-6140, Sigma Chemical Co, Saint Louis, MO, USA) or N-cadherin (1:50, #C-3865, Sigma Chemical Co, Saint Louis, MO, USA) for 1 h at 37 °C. Cells were washed and incubated for 1 h at 37 °C with secondary antibodies Alexa Fluor 546 or Alexa Fluor 488 (1:100), all purchased from Thermo Fischer Scientific. Nuclei were labeled with 0.1 μg/mL DAPI (Thermo Fisher Scientific) or NucSpot (1:500, Biotium, Hayward, CA, USA) for 5 min. Slides were mounted in ProLong Gold antifade reagent (Molecular Probes, Eugene, OR, USA) and examined in an Axiovert 100 inverted microscope (Carl Zeiss, Oberkochen, Germany). Images were acquired with an Olympus DP71 digital camera (Olympus, Shinjuku City, Japan). The overall fluorescence intensity was quantified using the ImageJ software (NIH, Bethesda, MD, USA), and results were expressed as a percentage, considering untreated cells as 100%.