96w20idf pet

Normalized transcellular electrical resistance (TER) was measured by electric cell-substrate impedance sensing [ECIS^®Zθ (theta)] instrument (Applied Biophysics Inc, Troy, NY, USA) as previously described (35 (link), 38 (link), 39 (link)). Briefly, HRECs were grown in 96-wells electrode arrays (catalog # 96W20idf PET, Applied Biophysics Inc.) coated with 100 µM cysteine and 0.02% gelatin. After confluence, cells were serum starved for 24 h and then treated with various treatments (BMP2 in the presence or absence of various inhibitors as above). For high glucose treatment, we used conditioned media (CM) that were collected from NG or HG-treated HRECs for 5 days. Fresh HRECs, then were subjected to these CM with or without various inhibitors (LDN1, LDN2, or noggin). TER was measured independently in each well over the time course of the experiment (4–5 days). Resistance values were normalized as the ratio of measured resistance to baseline resistance (normalized resistance) and plotted as a function of time.

The cell attachment behavior of the cells was analyzed real-time using electrical impedance spectroscopy (ECIS), a non-invasive technique that measures the impedance across gold electrodes at the bottom of tissue culture wells, using frequencies of alternating current^{41 (link),42 (link)}. Cells were plated in a 96-well ECIS array (Applied Biophysics, 96W20idf PET, Troy, NY) similar to those plated for the endpoint toxicity assays. The change in resistance at frequencies ranging from 400 to 64,000 Hz was measured over time. Low-frequency impedance can be used to monitor the solution paths around the cells, and hence the layer’s cell-to-cell barrier functions^{42 (link)}. The addition of particles may complicate the impedance of the system. However, at a frequency of 4,000 Hz, the contribution of resistance through the cells was dominant and, at much higher frequencies (8,000 to 64,000 Hz), the contribution is primarily from the added particles, in this case, biomass extracts with medium^{40 (link),43 (link),44 (link)}. Hence, a frequency of 4,000 Hz was chosen to monitor cell growth characteristics. Please refer to the Supplementary Information for more details regarding data collection and analysis.

HRECs were grown in 96-well electrode arrays (96W20idf PET, Applied Biophysics Inc.) and the electric currents passing through the monolayers were measured independently in each chamber. HRECs were seeded at a density of 5 × 10⁴ cells/chamber. Once confluent (Capacitance <20nF), the cells were serum-starved and treated with Hcy (20 μM and 50 μM) for 24 hours. Transendothelial resistance (TER) was recorded over the experimental time course. Integrity of the endothelial monolayer was confirmed microscopically at the end of each experiment and also by a final TER measurement. Resistance values for each chamber were normalized as the ratio of measured resistance to baseline resistance (normalized resistance).

Endothelial barrier function analysis was performed with impedance‐based cell monitoring using the electric cell‐substrate impedance sensing system (ECIS Zθ, Applied Biophysics). ECIS plates (96W20idf PET, Applied Biophysics, Troy, NY) were pretreated with 10 mM l‐cystein and coated with 1% gelatin. Baseline resistance was measured over ≈1 h after which endothelial cells were added to the plate. Multiple frequency/time mode was used for the real‐time assessment of the barrier and monolayer confluence. After ≈24 h when a stable barrier was formed, endothelial cells were stimulated with 500 ng/ml of recombinant EPHB2 (5189‐B2, R&D Systems, Minneapolis, MN, USA).