200 µL of solution (PA-YK-NO, PA-YK, or deionized (DI) water control) was added to a 48-well tissue culture plate (TCP) and allowed to self-assemble into nanomatrix coatings as previously described.22 (link)–23 (link) After drying, the treated wells were washed with DI water and UV sterilized for 2 hours. Human umbilical vein endothelial cells (HUVECs) were seeded at 15,000 cells/cm2. After 20 hours of culture, HUVECs were incubated with or without TNF-α (10 ng/mL) for 4 hours to stimulate inflammatory conditions. The HUVECs were then washed twice with warm, sterile PBS. U937 monocytes labeled with CellTracker Green CMFDA (Life Technologies) were then seeded on the HUVECs at a monocyte/endothelial cell ratio of 6:1. After 4 hours, unbound cells and media were aspirated from each well for unbound fraction determination. HUVECs were washed twice with warm sterile phosphate buffered saline (PBS) to remove loosely bound monocytes. Monocyte adhesion was analyzed using Nikon NIS Elements imaging software (Melville, NY) and a BioTek Synergy H1 microplate reader (BioTek Instruments, Inc.) at excitation and emission wavelengths of 485 and 528 nm, respectively.
Nis elements imaging software
Manufactured by Agilent Technologies
Sourced in United States
NIS Elements is an advanced imaging software platform developed by Agilent Technologies. It provides a comprehensive suite of tools for image acquisition, processing, analysis, and management across a wide range of microscopy techniques and applications.
Automatically generated - may contain errors
2 protocols using nis elements imaging software
1
Nanomatrix Modulation of Endothelial-Monocyte Interactions
2
Adipogenic and Osteogenic Differentiation of BMMSCs and SFMSCs
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Roughly, 2.0 × 105 BMMSCs and SFMSCs from each donor (n = 5) were induced to adipogenesis and osteogenesis. Cells were seeded in tissue culture dishes in the basal medium, with the medium change every 2–3 days. At 70–80% confluency, cells were induced to differentiate into each of the two lineages using lineage-specific differentiation media. Adipogenic differentiation was induced in complete growth medium supplemented with 1 µmol/l dexamethasone (BioTek, Winooski, VT, USA), 10 µg/ml recombinant human insulin (BioTek, Winooski, VT, USA), 0.5 mmol/l 3-isobutyl-1-methylxanthine (BioTek, Winooski, VT, USA), 15% rabbit serum (BioTek, Winooski, VT, USA), and 20 µmol/l indomethacin (BioTek, Winooski, VT, USA). Osteogenic differentiation was induced in complete growth medium supplemented with 100 nmol/l dexamethasone, 10 mmol/l β-glycerophosphate (BioTek, Winooski, VT, USA), and 0.25 mmol/l ascorbic acid (BioTek, Winooski, VT, USA). Adipogenic and osteogenic differentiation was confirmed at day 7 and 14 using 0.5% Oil Red O staining and 2% Alizarin Red S staining (Fisher Scientific, Pittsburgh, PA, USA), respectively. Stained cells were visualized and photographed using Nikon DS-Fi2 connected to a Zeiss microscope and evaluated using the NIS Elements imaging software (BioTek, Winooski, VT, USA).
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