Wallac victor

Intestinal integrity was determined by FITC-dextran assay^{61 (link),62 (link)}. Briefly, 6 μl/g body weight of FITC-conjugated dextran (100 mg/ml in PBS; 4.4 kDa FITC-dextran: FD4, Sigma-Aldrich) was administered to each mouse by intragastric administration. After 4 h, blood was collected from the retro-orbital plexus under pentobarbital anesthesia and centrifuged (10,000 × g at 4 °C) for 10 min. Diluted serum was added to a 96-well microplate in duplicate. The concentration of FITC in serum was determined by Spectro photo-fluorometry (Wallac Victor; Perkin-Elmer Life Sciences) with an excitation of 485 nm (20 nm bandwidth) and an emission wavelength of 528 nm (20 nm bandwidth) using serially diluted samples of the FITC-dextran marker as standard.

To assess oxidative stress, a fluorogenic probe (CellROX^® Green Reagent, Life Technologies, Vic Australia) designed to reliably measure reactive oxygen species (ROS) in live cells was used. The cell-permeable reagents are non-fluorescent while in a reduced state and upon oxidation exhibit a strong fluorogenic signal. IMR-32, IMR-32-CisRes and MRC-5 cells were plated in black 96-well plates with transparent bottoms at 10⁴ cells/well. Next day, cells were incubated for 5 h with Dextran-Catechin, prior to washing in PBS and measuring oxidative stress according to manufacturer's instructions using a fluorometer (Wallac VICTOR, PerkinElmer, Massachusetts, USA).

Phosphatase (PP1A/PP2) assay was performed with a colorimetric assay following the manufacturer’s instructions (Biozol, Eching, Germany). Phosphatases were incubated with ABT-737 and TQ and the reaction was started by 200 μM threonine phosphopeptide. After 15 min, reaction was stopped by Biomol Green (ENZO Life Sciences). After 30 min at RT, colour intensity was measured with absorbance at 600 nM in Wallac Victor (Perkin Elmer, Hamburg, Germany).

Equal number (4 X 10⁵) of VSMC infected with scrambled or ARF6 shRNA lentiviruses (day 3 of infection) were reseeded and serum starved for 48h (0.5%FBS). Cells were then stimulated or not with Ang II (100 nM) for 24, 48 or 72h. For each indicated time point, cells were stained using trypan blue, and live cells were manually counted using a hemocytometer. Cell proliferation was also measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. VSMC were cultured in 96-well plates (3 X 10³ cells/well), serum starved for 48h (0.5% FBS) and then stimulated or not with Ang II (100 nM). After 3 days, 25 μl of MTT (5 mg/ml) was added to the culture medium and cells were incubated for an additional 2h at 37°C before being solubilized in 20% SDS/ 50% dimethylformamide solution overnight. Absorbance was measured at 570 nm with a reference wavelength at 450 nm using the microplate reader (Wallac Victor; Perkin Elmer, MA, USA). For ARF6 knock down experiments, VSMC were seeded in the 96-well plates at the third day of infection with scrambled or ARF6 shRNA lentiviruses. DMSO vehicle, DPI, AG1478 or ML171 was added to medium 8h before Ang II stimulation. For Rac1T¹⁷N overexpression experiments, cells were cultured in 96-well plate 24h after plasmids transfection.

Migration of the lymphoblastic cell line Jurkat cells in response to CXCL12 or tagged-CXCL12 was evaluated using a transwell system. Briefly, cells were labelled with the intracellular fluorescent dye, calcein-AM (BD Biosciences), at 1 µM for 30 minutes at 37°C. Following labelling, cells were washed twice with PBS, and 2×10⁵ labelled cells in 100 µL of RPMI medium supplemented with 20 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) and 1% human AB serum were added to the upper chamber of a 96-well, 5-µm pore polycarbonate Transwell culture insert (Corning, Sigma). The same media (250 µL) containing 0 to 300 nM chemokine (either WT CXCL12, tagged-CXCL12, WT CCL5 or tagged-CCL5) was placed in the lower chamber. After 3 hours at 37°C in humidified air with 5% CO₂, fluorescence of cells that had been chemoattracted through the filter to the lower wells was measured using a fluorescence plate reader (excitation filter 485 nm and emission filter 535 nm) (Wallac Victor, Perkin Elmer).