Buffer was collected at specific time points (1, 3, 6, 24, 72, 144, 168 h) and stored at 4 °C until incubated with cells.
Buffer was collected at the indicated times and incubated with 300 μl of 3 mM 4-nitrophenyl N-acetyl-β D-glucosaminide (N9376, Sigma Aldrich) for 30 min at 37°C in 0.1 M citrate buffer (0.1 M sodium citrate, 0.1 M citric acid, pH 4.5).
Samples (300 μl) of serosal buffer were collected at 0, 30, 60 and 90 minutes after luminal addition of HRP, and were replaced with Krebs buffer.
Briefly, 300 µL samples of buffer were collected at the beginning and end of relaxation protocols in experiments with ACh or CPA (corresponding to AA/BH4 incubation times of 30 and 60 min).
At specific time points (1, 3, 6, 24, 72, 144, 168 h), buffer was collected and replaced with fresh buffer.
Storm water runoff was collected at 10-m intervals in three buffer types (native grass, fallow cropland, and Conservation Reserve Program).
Serosal buffer aliquots were collected at 0, 5, 15, 30 and 60 min after FITC addition and replaced with identical PBS amount.
Buffer solutions were collected at specific time points (0.5, 1, 3, 6, 24, 72, 144, 168 h) and replaced with an equal volume of fresh DMEM.
Purified GAGs were injected onto the column (1mL/minute) and washed with 5 volumes of binding buffer to remove unbound molecules before bound GAGs were eluted using a continuous salt gradient (0 1M NaCl in binding buffer), 1.5mL fractions were collected at a flow rate of 1mL/min and elution measured at an absorbance wavelength of 256nm.
The concentrate was applied to Sephadex G-150 column (70 cm × 1.8 cm) pre-equilibrated with 10 mM acetate buffer (pH 4.8) and the fraction were collected at a flow rate of 0.4 ml min−1.
This concentrated fraction was applied to Sephacryl S-200 column (1.5 110 cm) previously equilibrated with 10 mM glycine NaOH buffer (pH 8.0) and the fractions were collected at a flow rate of 0.2 mL min−1.
