Nitrate and nitrite levels produced by HUVEC after placental explant supernatant incubation for 24 h were assessed in HUVEC supernatant using a high-performance liquid chromatography (HPLC) system (ENO-20; Eicom, USA) as previously described (18 (link)). The ENO-20's high sensitivity and specificity were accomplished with the combination of a diazo coupling method and chromatography. The level of the diazo compound was measured by absorbance at 540 nm using a Spectramax iD3 multi-mode microplate reader (Hidex, Lablogic Systems, UK).
A total of 5×104 HUVEC per well were plated onto a black 96-well plate (Sigma-Aldrich). After 24 h of incubation with placental explant supernatants, the cells were washed with 95 µL of PBS and incubated for 30 min at 37°C. After this period, cells were loaded with 5 µL of DAF-FM™ (10 µM) (Sigma-Aldrich) and read for 60 min in 37°C using Spectramax iD3. The fluorescence signal was measured in a microplate reader (excitation 495 nm, emission 535 nm) and is reported as arbitrary units.
A total of 5×104 HUVEC per well were plated onto a black 96-well plate (Sigma-Aldrich). After 24 h of incubation with placental explant supernatants, the cells were washed with 95 µL of PBS and incubated for 30 min at 37°C. After this period, cells were loaded with 5 µL of DAF-FM™ (10 µM) (Sigma-Aldrich) and read for 60 min in 37°C using Spectramax iD3. The fluorescence signal was measured in a microplate reader (excitation 495 nm, emission 535 nm) and is reported as arbitrary units.
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