Mithras lb 940

Egg PC liposomes (extruded to 100 nm, 1 mM), STY-BODIPY (1 μM), and cholesterol (40 μM), desmosterol (40 μM), 7-DHC (40 μM), or vehicle (MβCD) were vortexed in PBS (10 mM, 150 mM NaCl, pH7.4), then 200 μl aliquots were incubated in a black 96-well plates (polypropylene, Nunc) for 20 min at 37 °C. DTUN (200 mM in EtOH) was added to the aliquots. The plate was mixed for 5 min and kinetic data of STY-BODIPY_OX was acquired at 488 nm excitation and 518 nm emission by Mithras LB940 microplate reader (Berthold Technologies)^{29 (link)}.

Metabolic activity of aortic tissue was assessed using an assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) by viable cells with CellTiter 96^® AQ_ueous One Solution Assay (Promega). Tissue punches were incubated in DMEM overnight at 37 °C. Medium was exchanged before MTS assay was performed according to the manufacturer’s protocol. Absorbance at the 490 nm wavelength was measured (SpectraMax 340PC; Molecular Devices, Sunnyvale, CA, USA).
Necrotic cells or cells with membrane damage in aortic tissue were determined by a lactate dehydrogenase (LDH) release assay (CytoTox-ONE™ Homogeneous Membrane Integrity Assay; Promega). Tissue punches were incubated in DMEM for 1 h at 37 °C before assay was performed according to the manufacturer’s protocol. Fluorescence was read with excitation at 560 nm and emission at 590 nm (Infinite 200 PRO, Tecan, Männedorf, Switzerland).
Caspase 3/7 activity was determined using Caspase-Glo^® 3/7 assay (Promega). Tissue punches were incubated in DMEM for 1 h at 37 °C before assay was performed according to the manufacturer’s protocol. After 1 h incubation, luminescence was measured (Mithras LB 940, Berthold Technologies, Bad Wildbad, Germany).

5.10³ cells per well were seeded into 96-well plates and were treated for 24 h with DEET. After incubation, the plate was shacked for 15 s. The supernatant with non-adherent cells was removed by three washes with washing buffer (0.1% BSA in medium without serum). Attached cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were rinsed two times with washing buffer, stained with crystal violet (1 mg.mL⁻¹ in 2% of ethanol) for 10 min at room temperature and extensively washed with distilled water. Then, SDS 2% was added and incubated for 30 min at room temperature. Absorbance was then evaluated using a Mithras LB940 multimode microplate reader at 550 nm (Berthold Technologies).

The Gi, Gq, Go, Gs, and Gz dissociation BRET probes were from the TRUPATH kit, which was a gift from Bryan Roth (Addgene kit #1000000163 (31 (link))). HEK293 cells were cotransfected with BmOR3 along with specific G protein BRET probes. After 24 h, the cells were reseeded in 96-well microplates and incubated for another 24 h. The cells were washed twice with Hank's balanced salt solution and stimulated with bombykal at different concentrations for 2 min. The light emission of Rluc8 (400 nm) and GFP10 (510 nm) was measured after the addition of the substrate coelenterazine 400a (5 μM) using a Mithras LB 940 multimode reader (Berthold Technologies). The BRET signal was calculated as the ratio of light emission at 510/400 nm.

SIV-specific neautralizing antibodies (Nabs) were evaluated using the TZM-bl neutralization assay as previously described [36 (link)]. Briefly, serial diluted heat-inactivated serum samples were mixed with 50 TCID₅₀ viruses and incubated at 37°C for 1 hour. After incubation, 10⁴ TZM-bl cells were added to each well with DEAE-dextran at a final concentration of 12.5 μg /ml to enhance virus infectivity. After 40 hours of culture at 5% CO2 and 37°C, luciferase activity was measured with the Luciferase assay kit (Promega) and read on Mithras LB940 (Berthold technologies). Average relative luminescence units (RLU) of cell controls were subtracted as background and the percent inhibition plotted for each serum dilution.

The biocompatibility was assayed according to the DIN EN ISO 10993-5 and -12 guidelines using the cleavage of the tetrazolium salt WST-1 (4-(3-(4-Iodophenyl)-2-(4-nitrophenyl) -2H-5-tetrazolio)-1,3-benzene disulfonate) to formazan. Ten independently manufactured samples with LightPLAS coating were each tested in triplicate (n = 10). The samples were first washed with 1 mL RPMI 1640 medium. Then, the extraction was carried out in 1 mL RPMI 1640 medium per sample at 37 °C, 72 h prior to the application of extracts on cells. L929 cells (100 µL cell suspension, 10⁵ cells/mL) were seeded into a 96-well plate and incubated for 24 h at 37 °C, 5% CO₂. An amount of 100 µL supernatant was substituted with 100 µL extract. After an additional 24 h of incubation, the solutions were removed from the wells and substituted with 150 µL of the proliferation reactant WST-1 (10 µL WST-1/mL RPMI). After 1 h of incubation with WST-1, 100 µL of each well was transferred to a new plate and the absorbance was measured at 450 nm with a multimode reader (Mithras LB 940, Berthold Technologies, Bad Wildbad, Germany). Untreated cells were used as negative control (biocompatible) and a treatment with 10% hydroxyethyl methacrylate (HEMA) was used as positive control (cytotoxic).

LV and RV C-MSC were plated at a concentration of 30,000 cells/cm² in the growth medium with a reduced amount of FBS (2%; see section “C-MSC Isolation and Culture”) for 5 days, without medium change. The collagen production and myofibroblast differentiation were assessed through Sircol collagen analysis (Biocolor Life Science Assays), Western Blot, and qRT-PCR. Sircol Collagen Assay was performed on LV and RV C-MSC lysates and supernatants, after their collection in low-protein-binding tubes. The cellular lysates underwent collagen isolation and concentration step overnight. Both C-MSC lysates and supernatants were then mixed with 1 ml of Sircol Dye Reagent at RT for 30 min to ensure the precipitation of collagen. The obtained pellet was dissolved in Alkali Reagent, and the amount of collagen was determined at 540 nm using a microplate reader (Mithras LB 940; Berthold Technologies) and calculated based on a standard curve of soluble collagen.