Clariostar spectrophotometer

The ratio of reduced (GSH) to oxidised (GSSG) glutathione was determined using a commercial assay (GSH : GSSG-Glo assay, Promega, Southampton, UK) according to the manufacturer's instructions, with cells plated at 200,000 cells/cm² on black-walled 96-well plates. A CLARIOstar spectrophotometer (BMG Labtech, Germany) was used to measure relative luminescence with comparison to a total glutathione standard curve (0.25 μM-16 μM).

The total content of phenolic compounds was assessed using the Folin–Ciocalteu method. Briefly, Salicornia extract stocks (20 mg/mL in 20:80 v/v ethanol:water) were diluted to 1 mg/mL in water. Then, 30 μL of each sample (1 mg/mL) or standard (Gallic acid) were mixed in triplicates with 30 μL of Folin–Ciocalteu reagent (1:10 dilution in water) in a 96-well plate and incubated at room temperature for 5 min. Finally, 5% sodium carbonate (w/v, 240 μL) was added and the plate was incubated for 1.5 h at 30 °C. Absorbance was measured at 760 nm using a CLARIOstar spectrophotometer (BMG Labtech, Ortenberg, Germany). Total phenolic content was calculated using a Gallic acid calibration curve within a range of 0–100 μg/mL. Results were expressed as mg Gallic acid equivalents (GAE)/g of dry extract.

The LDH activity was evaluated with the PicoProbe LDH Cytotoxicity Fluorometric Assay Kit (BioVision). The LDH was measured using 10 μL of the cell supernatant to detect the LDH released from the cells after membrane damage, mixed with the buffer solution and PicoProbe up to 200 μL as in the manufacturer's protocol. The mixture was incubated in a black fluorescence 96 well plate and read at λEx/Em = 535/587 nm. The fluorescence intensity indicates the intensity of the LDH activity. The Fluorometric measurements were performed using CLARIOstar spectrophotometer (BMG Labtech). The results are expressed as mean values and the experimental groups were compared to the control group, DAMI Luc2 cells without CAR T cells in co‐culture.

Mice (n = 5–6 per group) were injected intraperitoneally (i.p.) with 1.8 mg/kg body weight TMAO in 100 μl saline vehicle, a dose calculated to approximate human circulating TMAO levels [42 (link)], followed 2 h, 6 h or 24 h later by assessment of Evans blue extravasation as described below. Alternatively, mice were injected i.p. with 3 mg/kg body weight LPS or 100 μl 0.9% saline vehicle, followed 2 h later by i.p. injection of either 1.8 mg/kg body weight TMAO or 100 μl 0.9% saline vehicle for assessment of Evans blue extravasation 2 h later. In both experiments, one hour before assessment animals were injected i.p. with 100 μl of a 2% (w/v) solution of Evans blue dye in 0.9% saline (Sigma–Aldrich Ltd, Poole, UK). Dye was permitted to circulate for 1 h before animals were transcardially perfused with 0.9% saline at 4 °C to remove circulating dye. Brains were removed, bisected and homogenised by maceration in 0.9% saline. Suspended macromolecules were precipitated by incubation with 60% trichloroacetic acid, and dye content of resulting supernatants was detected using a CLARIOstar spectrophotometer (BMG Labtech GmbH, Germany) alongside a standard curve of defined concentrations of Evans blue in the same buffer. Brain Evans blue content was expressed as μg of dye per mg of brain tissue, normalised to circulating plasma concentrations.

hCMEC/D3 cells were grown on 6-well plates coated with calf-skin collagen (Sigma–Aldrich, Gillingham, UK) and collected in TRIzol (Thermo-Fisher Scientific, UK) as described previously [2 (link)]. Total RNA was extracted using a TRIzol Plus RNA purification kit (Thermo-Fisher Scientific, UK) and quantified using a CLARIOstar spectrophotometer equipped with an LVis microplate (BMG Labtech GmbH, Germany).
Hybridization experiments were performed by Macrogen Inc. (Seoul, Republic of Korea) using Illumina HumanHT-12 v4.0 Expression BeadChips (Illumina Inc., San Diego, CA) as described previously [2 (link)].

The potential for pCG-induced cytotoxicity was assessed using the MTT assay. Briefly, cells were treated with pCG for 24 h (0.1, 1, 10, 100 µM), prior to administration of MTT at 500  μg/ml. Cells were incubated at 37°C for 2 h, medium was removed and resulting crystals were solubilized by incubation for 2 minutes in dimethyl sulfoxide. Absorbance was read at 540 nm using a CLARIOstar spectrophotometer (BMG Labtech, Ortenberg, Germany), with a reference wavelength at 570  nm.

Mice (n = 5–6 per group) were injected i.p. with 1 mg/kg body weight pCG in 100 µl saline vehicle, a dose calculated to approximately double circulating concentrations,^{18 (link)} followed 2 h or 6 h later by assessment of Evans blue extravasation. One hour before assessment animals were injected i.p. with 100 µl of a 2% (w/v) solution of Evans blue dye in 0.9% saline. Dye was permitted to circulate for 1  h before animals were transcardially perfused with 0.9% saline at 4°C to remove dye remaining in the vasculature. Blood samples were allowed to coagulate at 37°C for 15 minutes prior to centrifugation at 800 g for 10 minutes to separate serum. Brains were removed and homogenized by maceration in 0.1 M phosphate-buffered saline. Suspended macromolecules were precipitated by incubation with 60% trichloroacetic acid, and dye content of resulting supernatants was detected using a CLARIOstar spectrophotometer (BMG Labtech GmbH, Germany) alongside a standard curve of defined concentrations of Evans blue in the same buffer. Brain Evans’ blue content was expressed as µg of dye per mg of brain tissue, normalized to circulating serum concentrations.