Cellsens imaging software

Becton Dickinson (BD)-BioCoat Angiogenesis System-EC tube formation 96-well plates (BD Biosciences, Bedford, MA, USA) were used. Cells from each group were plated at 2 × 10⁵ in 50 µL media in each well. The plates were incubated for 16–18 h at 37 °C and were then labelled with BD calcein AM Fluorescent dye. The plates were imaged at 4× magnification (scale bar is 200 µm) using an Olympus BX53 microscope, DP72 digital camera, and CellSens imaging software (Olympus, Center Valley, PA, USA). Tube formation assays were conducted in three 96-well plates, one in Nx, one in Hx, and one in IH. In each plate, 32 wells were treated with saline, 32 were treated with low dose bumetanide (0.05 µg/mL), and 32 were treated with high-dose bumetanide (0.2 µg/mL). Quantitative analysis of the number of tubes formed was conducted using the count and measure tool from the CellSens imaging software (Olympus). Only fully formed tubes with complete branching polygons forming a central vacuole were counted. Data are presented in Table 1.

Nodose ganglia and whole brains (n=4/group) were immersed in 4% paraformaldehyde/PB for 24 h at 4 °C, incubated for 24 h in PB containing 20% sucrose, quickly frozen on dry ice, and cut into 8-μm slices with a cryostat at −20 °C. Sections blocked for 5 min in protein-block serum-free solution (Dako, Carpinteria, CA, USA) were incubated overnight at 4 °C with rabbit anti-IBA1 (1:10 000; Wako Pure Chemicals), rat anti-CD11b (1:50; AbD Serotec, Oxford, UK), and rat anti-CD86 (1:100; Abcam, Cambridge, UK). Immunofluorescence was performed with a combination of Alexa Fluor 488-labeled anti-rabbit secondary antibody or Alexa Fluor 594-labeled anti-rat secondary antibody (both 1:400; Invitrogen). Images were captured on an OLYMPUS AX-7 fluorescence microscope (Olympus). Cells immunostained with IBA1, CD11b, or CD86 antibody were counted manually with Olympus cellSens Imaging Software (Olympus). Quantitation was performed in a blinded fashion.

Matrigel (BD Biosciences, NJ, USA) was added at 50 µL/well of a 96-well plate and incubated for 30 min in a humidified incubator at 37 °C allowing the Matrigel to polymerize. B16-F10 and HBME cells (2 × 10⁴) were suspended with appropriate growth media and loaded on top of the Matrigel-coated wells in triplicates for each condition. Cells were incubated overnight at 37 °C, and pictures were acquired from five independent fields per well using Olympus cellSens imaging software (Olympus, Tokyo, Japan) to quantify the number of tubes in triplicate wells. The average value for each condition is the mean of three tube numbers taken from three separate wells. Cells transfected with empty pcDNA3 plasmid were considered as a control condition.

The wound healing assay utilized the wound healing assay kit (Abcam, UK) in accordance with manufacturer’s guidelines. In summary, the inserts were positioned in the plates to create a uniform wound field in a specific direction, ensuring secure contact with the plate well bottom. A 500 μL HOB cell suspension (7.5x10⁴/ well) was gently added through the insert’s open end at the top, with an additional 250 μL on either side for optimal cell dispersion. The plates were then incubated in a cell culture incubator until a monolayer formed. Following incubation, the inserts were carefully removed, and the culture wells were washed with 1X PBS to eliminate dead cells and debris. HOB cells were treated accordingly as mentioned in Table 1. in complete DMEM /F12 media supplemented with 2.5% FBS. Subsequently, HOB cells were allowed to migrate for 24 h. Wound closure was observed using a light microscope, and images were captured both time zero and 24 h using Olympus cell Sens imaging software (Olympus Life Science, Tokyo, Japan). The percentage closure or migration rate of HOB cells into the wound field was then analyzed using Image J Software (National Institutes of Health, USA).

Bladders were fixed in 4% paraformaldehyde for 1 h, rinsed with PBS, dehydrated in a series of graded alcohol, and embedded in paraffin. Tissue sections (5 μm sections) were stained with hematoxylin and eosin (H&E) to evaluate bladder wall thickness, and Sirius Red staining was performed to visualize collagen deposition.
The bladder wall thickness was measured at 5 random locations to calculate the average thickness. Images of 5 randomly chosen areas from Sirius Red staining at 10 × magnification were taken using the Olympus cellSens Imaging software (Olympus Corporation, Tokyo, Japan). By using bright-field microscopy with or without polarization filter, the collagen fibers stained by Sirius Red appeared bright red (i.e., collagen I) and bright green (i.e., collagen III) in sharp contrast with the rest of the tissue remaining black. The collagen deposition in the smooth muscle layer was quantified by Image J at 20 × magnification from 5 randomly captured images. The collagen area was presented as the percentage of total tissue area.

Stably transfected B16-F10 clones, transiently transfected HBME cells, and stably transfected HBME cells with control plasmid or PAI-1 shRNA plasmid were then treated with B16-F10 empty pcDNA3 (control) or B16-F10 mixed clones (14 kDa hGH) conditioned media for 48 h and cultured in 6-well plates at 2.5 × 10⁵ cells/well seeding density. At 90% confluency, a linear wound was created by vertically scratching the confluent cell monolayer using a sterile 200 µL micropipette tip. Images were then captured at fixed positions along the wound at 0, 24, and 48 h using Olympus cellSens imaging software (Olympus, Tokyo, Japan). Pictures were successively analyzed for percent wound closure using ImageJ software. The average value for each condition is the mean of three measurements taken from three separate wells.