Tms inverted microscope

Cell migration was studied using a wound-healing assay. SKOV3 and HEY cells (100,000 per well) were seeded on 24-well plates containing 500 μl of RPMI medium. The cells were then washed 3 times with PBS, and a wound was generated by removing the cells in the center of the well with a sterile pipette tip. The detached cells were washed away with PBS. The cells were incubated in different experimental conditions for 6 to 24 h. Cell migration was studied by treating the cells with 100 ng/ml of leptin alone or with a 30 min pre-incubation with the leptin-neutralizing antibody (10 μg/ml), AG490 (50 μM), fasudil (10 μM), UO126 (10 μM) or LY294002 (10 μM). Three images were obtained along the wound with a Nikon TMS inverted microscope connected to a Nikon Coolpix 4500 camera (Nikon Instruments Inc.). Wound closure was quantified by measuring the area in pixels between the edges of the wound using a measurement tool in Adobe Photoshop^® with a grid superimposed on the image as a guide for the measurements. The wound width was normalized to 100% at 0 h for each treatment condition, and the data represent the percentage of wound closure.

We collected 50–250 mL of water samples with detritus and/or plant material from ponds or lakes in Michigan in 2019–2021 (see Table S2 in the supplemental material). For Lake Erie, seston was collected by boat with a plankton net (≥20 µm) deployed 1–3 m from the surface, after which the collected material was transferred to a 50 mL conical centrifuge tube maintained at in situ water temperature in the dark. The samples were transferred to University of Michigan and incubated for ~1 month, at 20°C, under LED lighting. Water samples were observed using a Nikon TMS Inverted microscope (Nikon, Tokyo, Japan) to detect fungi associated with algae, micro-invertebrates, and protists. Detected fungal cells were photographed using Moticam X Camera (Motic, Hong Kong, China) or Dino-Eye Edge S Eyepiece Camera (AnMo Electronic Corporation, Taipei, Taiwan) digital cameras. Representative images were edited and assembled into plates using Adobe Photoshop. The cells were isolated manually using a manually prepared drawn-out glass capillary pipette. The isolated cells were washed by serial transfer in small drops (more than five) of UV-sterilized water, transferred into 200 µL PCR tubes with 1–2 µL of water, and kept at −80°C until DNA extraction.

All tissue culture-based studies were conducted in a class II biosafety laminar flow hood under sterile conditions. Cells were propagated and maintained in 25 cm² tissue culture flasks containing 5 mL of complete medium (EMEM containing 10% (v/v) FBS) and antibiotics (100 U·mL⁻¹ penicillin, 100 μg/mL streptomycin) at 37 °C in a HEPA class 100 Steri-Cult CO₂ incubator (Thermo-Fisher Corporation, Waltham, MA, USA). Cells were monitored daily under a Nikon TMS inverted microscope (Nikon Corp, Tokyo, Japan). Cells were subcultured into multiwell plates for assays or cryopreserved.