Tristar2 lb 942

Cell viability was assessed with MTS reagent (Promega™, G358C, Charbonnières les Bains, France), in accordance with the manufacturer’s instructions. Cells were seeded into 96-well cell culture plates and incubated for 24 h at 37 °C in 5% CO₂. Next, treated with different concentrations of Baicalein (5 to 40 μM) and H₂O₂ (50 to 800 μM). Subsequently, the cells were incubated with 15 μL MTS reagent in DMEM at 37 °C for 2 h. Absorbance was measured at 490 nm, using a microplate reader (TriStar2-LB942, BERTHOLD, Bad Wildbad, Germany).

The cytotoxicity of the water-soluble fractions of the soil samples and the leachate samples was assessed with PrestoBlue. A total of 5000 (Caco-2, Hep-G2, IEC-6) or 1000 (A-549) cells/well were placed in 96-well black flat-bottom plates and incubated for 24 h at 37 °C in 5% CO₂. The next day, the medium was removed from the cells, and then dilutions of leachate samples and soil extracts were added. The final concentrations of the analysed soil extracts were (mg/mL): 3.13; 6.25; 12.5; 25; 50; 100, while for the leachate samples (mg/mL): 31.3; 62.5; 125; 250; 500. Cells in the culture medium were negative control. The samples were exposed for 72 h at 37 °C in an atmosphere of 5% CO₂. After this, the samples were aspirated, and PrestoBlue (10% solution in PBS) was added to each well and incubated for a further 2 h at 37 °C in 5% CO₂. A microplate reader (TriStar2 LB 942, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) was used to measure fluorescence (λ_ex 560 nm; λ_em 590 nm). IC₅₀ values were estimated from the resulting curves.

Neutralization assays with serum IgGs against the 12-strain global virus panel, were performed in 96-well plates following published protocols^{9 (link),90 (link)}. To this end, 12 HIV-1 pseudovirus strains were each mixed with 1:2 serial dilutions of purified IgG (1 mg/ml starting concentration, 8 dilutions) and incubated for 1 h at 37 °C. TZM-bl cells (RRID:CVCL_B478; ordered from the HIV Reagent Program, Cat-No. ARP-8129) were added (10⁴ per well) in growth medium (DMEM, Gibco; 10% heat-inactivated FBS, Sigma-Aldrich; 2 mM L-glutamine, Thermo Fisher; 1 mM sodium pyruvate, Gibco; 50 µg/ml gentamicin, Sigma-Aldrich; 25 mM HEPES, Biochrom) with DEAE-dextran at a final concentration of 10 µg/ml and incubated for 2 days. Equal amounts of Luciferin-containing lysis buffer (10 mM MgCl₂, 0.3 mM ATP, 0.5 mM Coenzyme A, 17 mM IGEPAL (all Sigma-Aldrich), and 1 mM D-Luciferin (GoldBio) in 200 mM Tris-HCL pH 7.8) was added and after 2 min incubation samples were resuspended and luminescence was measured with a luminometer (Berthold TriStar² LB942). For IC₅₀ determination, the background signal (non-infected TZM-bl cells) was subtracted and IgG concentrations resulting in a 50% RLU reduction compared to untreated virus control wells were determined by using murine leukemia virus (MuLV)-pseudotyped virus as a control for unspecific activity. All samples were tested in duplicates.

Luciferase assay was carried out using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, control or Lpin2 knockout RAW-D cells were transfected with pGL4.32[Luc2P/NF-kB-RE] firefly luciferase reporter and pRL-TK renilla luciferase expression plasmids (Promega) at a ratio of 50:1. At 24 h after transfection, cells were stimulated with 200 ng/mL LPS. After 4 h, cells were harvested for evaluating reporter activities. The firefly and renilla luciferase activities were measured in a multimode reader, TriStar2 LB942 (Berthold Technologies, Bad Wildbad, Germany). The values of firefly luciferase activities were normalized with that of renilla luciferase.

LY dye migration through the BBB monolayers was performed as previously described [25 (link), 26 (link)]. Briefly, Transwell^® inserts containing hCMEC/D3 monolayers were transferred in culture wells containing 1.5 mL of Hanks’ Buffered Salt Solution (HBSS) supplemented with 10 mM of HEPES buffer, 1 mM of sodium pyruvate and 50μM of LY (Sigma-Aldrich; catalog no. L0144). The culture medium inside the Transwell^® inserts was replaced with 500μL of HBSS buffer containing 50μM of LY. Cells were incubated at 37°C for 10 min. Permeable inserts were then transferred in culture well containing 1.5 mL of HBSS buffer and incubated at 37°C for 15 min. They were then transferred in culture well containing 1.5 mL of HBSS buffer and incubated at 37°C for 20 min. Concentrations of LY in the wells were determined using a fluorescent spectrophotometer (Berthold, TriStar² LB 942). The emission at 535 nm was measured with an excitation light at 485 nm. The endothelial permeability coefficient of LY was calculated in centimeters/min (cm/min), as previously described [30 (link)].

A total of 7.7 × 10³ cells/well were seeded in 96-well microtiter plates one day before treatment and treated as indicated. Subsequently, 10% CellTiter 96^® AQueous One solution containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) (Promega, Madison, WI, USA) was added and incubated for 1–2 h before measuring the absorbance at 492 nm in a TriStar² LB 942 microplate reader (Berthold Technologies GmbH & Co.KG, Bad Wildbad, Germany).