Cellsense imaging software

The cell capacity to migrate was evaluated by seeding parental or MTDH-edited cells (SUM-149, SUM-190, and MCF-10A) in two-well silicone inserts with a defined cell-free gap wound plate (Ibidi USA Inc., Madison, WI, USA). For SUM-149, 4 × 10⁴ cells/well were seeded, 8 × 10⁴ cells/well for SUM-190, and 1 × 10⁵ cells/well for MCF-10A. All cell lines were cultured in their respective complete culture medium for 24 h. After the incubation period, the insert was removed and the cells were allowed to migrate for 24 h at 37 °C as described by us in [74 (link)]. For MCF-10A cells, a complete culture medium with 2% HS was used to perform the assay. The cells were then fixed with 4% paraformaldehyde for 15 min, washed with 1X PBS, permeabilized with 0.1% Triton X-100 for 15 min at RT, and blocked with 1% BSA. Cells were stained for 1 h with a 1X rhodamine-phalloidin solution (Invitrogen^TM/Life Technologies) to visualize actin filaments (F-actin). After washing three times with 1X PBS, cells were incubated with 1 µg/mL of DAPI (Life Technologies) for nuclear staining. Cell migration was quantified by measuring the distance (µm) between the wound edges using Olympus CellSense Imaging Software (Center Valley, PA, USA) on micrographs at a magnification of 4×. Fluorescence images were obtained at a magnification of 20× using the Cytation 10 Confocal Imaging Reader (Agilent/BioTek).

On day 18 of gestation, 28 dams were euthanized using CO₂ inhalation to excise uterine horns and collect placental tissues and fetuses. Tissue samples were immediately placed in a 10% formalin solution for approximately 24 h. After 24 h, all tissue samples were rinsed with a PBS solution, transferred to a 70% ethanol solution, and stored at 4 °C until they were submitted for staining. Placental samples were embedded in paraffin using standard histological techniques, sectioned at a thickness of 4 µm, and stained with hematoxylin and eosin at the Comparative Pathology Shared Resource Laboratory (Masonic Cancer Center at the University of Minnesota, Minneapolis, MN, USA). Stained slides were evaluated by light microscopy using an Olympus BX53 Microscope (Center Valley, NJ, USA) at 4× power. CellSense imaging software (Version 1.18; Olympus, Center Valley, NJ, USA) was used to outline the total labyrinth area. The labyrinth area was determined in two separate tissue sections for each placenta. The average measurement of two sections was recorded as the area of the labyrinth. Measurements of 208 placentae were completed by one person who was blinded to treatments to reduce variation.

The scratch assay is a well-developed method to investigate the HCT116 cell migration in vitro. The cells were seeded at a density of about 1 × 10⁶ cells/well (10⁴ cells/cm², in 6-well plates) in complete medium at 37 °C and 5% CO₂ (v/v), and grown for 24 h to allow them to reach about 90% confluence. Scratches were created mechanically with a sterile pipette tip (Ø = 0.1 mm) on cell monolayer. We were careful to produce uniformly sized wounds of approximately 0.5 mm, and debris was removed from the culture and cells were then cultured with fresh medium. The size of the gap at a selected position was measured using the cell Sense Imaging software (Olympus, Hamburg, Germany) at the starting point of the experiment (0 h), after 24 h and up to 48 h. The ratio of the gap size at certain time points to the gap size at 0 h was calculated and replicates were averaged. Scratch closure was assessed every 2 h using an inverted microscope (Leica DM IL D-35578 Wetzlar, Germany) at a magnification of x10 and photographed with a Colour View II digital camera to measuring the remaining cell-free area in triplicates wells. The results were expressed as percentage of the cell-free area at T 24 h or T 48 h compared to T₀ and represented as mean ± SD of 3 independent experiments.

Completed wafers were imaged using a digital microscope with a zoom lens (Sciencescope International, Chino, CA, USA) to inspect wafer quality and features. PDMS replicas and cross-section samples were imaged using an inverted microscope (Olympus IX-83, Olympus America Inc., Lombard, IL, USA) fitted with a high-resolution sCMOS camera (Zyla 5.5, Andor, Concord, MA, USA). The images were later analyzed and measured using CellSense imaging software (Olympus America Inc., Lombard, IL, USA).

To mimic conventional, well-accepted methods of measuring adipocyte number and size, we used an Olympus BX43 microscope with an Olympus DP27 camera to obtain one representative micrograph of each sample at 10x magnification (cellSense imaging software; Olympus Corporation, Japan). We obtained manual counts of the number of adipocytes in each image identifiable by a trained observer. We also loaded the images into ImageJ for automated counting using the MRI Adipocyte Tools plugin (See Supplementary Methods for step-by-step details). We first defined our desired adipocyte size range (500–20,000 µm²), then removed background by setting the number of dilates, or connections among defined adipocytes, to 10. We segmented adipocytes within the size specifications using ‘Percentile’ thresholding, followed by the ‘Simple Segmentation’ command to count the number of cells within an image. A board-certified pathologist reviewed all of the slides and associated analyses to confirm appropriate identification of adipocytes. We then used the ROI Manager to determine the area of each detected adipocyte. This generated a Results window from which we recorded the area of each adipocyte and the mean adipocyte area, minimum adipocyte size, and maximum adipocyte size within each image.

The microchannel was placed on the stage of an inverted epi-fluorescent microscope (IX83, Olympus America, Center Valley, PA, USA). Fluorescent images of particle flow inside the microchannel were acquired using a high-resolution sCMOS camera (Andor Zyla 5.5, Oxford Instruments, Santa Barbara, CA, USA) mounted on the microscope along with the CellSense imaging software (Olympus America, Center Valley, PA USA). At least 150 fluorescent images were acquired at the channel outlet for every flow rate. The fluorescent images were stacked to generate composite micrographs of particle streaks. Bright-field images were obtained using a high-speed camera (Photron Mini AX200, Photron USA. Inc., San Diego, CA, USA). All images were analyzed using ImageJ^® (NIH, Bethesda, MD, USA).