Modulus microplate

Viral stocks of the R5‐tropic infectious molecular clone NL‐LucR. T2A‐BaL.ecto (HIV‐1_{BaL‐Renilla})⁶⁶ were prepared as previously described.⁴⁰, ⁶⁷ Viral titres were determined as previously described⁴⁰, ⁶⁶ and calculated as log infectious units (IU)/ml.⁶⁸Non‐activated bbPBMCs were seeded at 4 × 10⁶ and treated in vitro with the indicated ligands for 2 or 7 days. bbPBMCs were then infected using 10 IU/ml HIV‐1_{BaL‐Renilla} or the equivalent volume of virus control for 2 h at 37°C, and subsequently washed three times in 1 X PBS containing 1% cs‐FCS, resuspended in hormone‐free full RPMI containing IL‐2 and incubated for a further 5 days. PBMCs were pelleted by centrifugation then harvested either for Renilla luciferase expression (infection) using Renilla luciferin according to the manufacturer's specifications (Promega, USA), measured on a luminometer (Modulus microplate, Turner Biosystems, USA) or for cell viability using the MTT assay, measured on a spectrophotometer (Thermo Scientific, USA) at 595 nm. Infection was calculated as RLU of each quadruplicate divided by the average absorbance at 595 nm of the four quadruplicate wells (RLU/ave MTT). Relative infection was determined by setting the vehicle control (EtOH) in the presence of virus to 100% or as indicated in the figure legend. Archived hPBMCs were not used for infection experiments due to low sample availability.

Initial viral stocks were prepared as previously described [30 (link)] with a few modifications. HEK293T cells were seeded at a density of 4 X 10⁶ cells in a 10 cm² plate in full DMEM supplemented with 25 mM HEPES buffer (Lonza, Germany) at 37°C in a water-jacketed incubator (90% humidity and 5% CO₂). The next day, media was replaced and cells were transfected with 12 μg HIV-1_BaL-Renilla or a control (DMEM) using X-tremeGENE 9 DNA transfection reagent (Roche Applied Science, South Africa) according to the manufacturer’s specifications. Cells were incubated for 48 hours at 37°C, the medium was passed through a 0.22 μM filter and charcoal-stripped (c-s) FCS (Thermo Scientific, USA) was added to a final concentration of 12.5%. The viral stocks were aliquoted and stored at -80°C until use. Virus titres were determined using the TZM-bl assay as previously described [29 (link)]. Cells were harvested 72 hours later with 120 μL Bright-Glo luciferase lysis buffer (Promega, USA). Fluorescence was determined on a luminometer (Modulus Microplate, Promega, USA), where relative light units (RLU) were measured for each well. The titre of the virus stock was determined using the Reed Muench method and expressed as log infectious units (IU)/mL [31 ].

Initial viral stocks were prepared as previously described^{68 (link)} with a few modifications. HEK293T cells were seeded at a density of 4 × 10⁶ cells/10 cm² plate in full DMEM supplemented with 25 mM HEPES buffer (Lonza, Germany) at 37 °C in a water jacket incubator (90% humidity and 5% CO₂). The next day, media was replaced, and cells were transfected with 12 μg HIV-1_pNL4.3, HIV-1_BaL-Renilla or a control (DMEM) using X-tremeGENE 9 DNA transfection reagent (Roche Applied Science, South Africa) according to the manufacturer’s specifications. Cells were incubated for 48 hr at 37 °C, the medium was passed through a 0.22 μM filter and charcoal-stripped (c-s) FCS (Thermo Scientific, USA) was added to a final concentration of 12.5%. The viral stocks were aliquoted and stored at −80 °C until use. Virus titres were determined using the TZM-bl assay as previously described^{67 (link)}. Cells were harvested 72 hr later with 120 μL Bright-Glo luciferase lysis buffer (Promega, USA). Fluorescence was determined on a luminometer (Modulus Microplate, Promega, USA), where relative light units were measured for each well. The titre of the virus stock was determined using the Reed Muench method and expressed as log infectious units (IU)/mL⁶⁹ .

TZM-bls were seeded at a concentration of 5 X 10⁴ cells/mL in a 96-well flat bottomed culture plate in full DMEM. The following day the TZM-bl cells were either stimulated with hormone or maraviroc (MVC) for 24 hours in triplicate. Cells were then infected with 20 IU/mL HIV-1_{BaL_Renilla} and were harvested 48 hours later with Bright-Glo luciferase lysis buffer (Promega, USA). Luminescence was determined on a luminometer (Modulus Microplate, Promega, USA), where relative light units were measured for each well. Viability was measured using the MTT assay and measured on a spectrophotometer (Thermo Scientific, USA) at 595 nm. Luciferase readings were normalized to MTT values (RLU/MTT). Relative infection was calculated by setting the vehicle control (EtOH) to 100% relative infection.

MDA was quantified by derivatization with TBARS as previously published^{25 (link)}. Brain tissues (0.4 mg) or plasma (200 μL) were treated with 4% butylhydroxytoluene (MP Biomedicals) to prevent further oxidation. Phosphate buffer (pH 7.4) was added and the samples were incubated for 30 min at 37 °C under constant agitation. TBARS (0.8%) and 20% acetic acid (pH 3.5) were added and the mixture was incubated for 1 h at 100 °C. Placing the mixture in ice stopped the reaction; afterwards 2% KCl and 5 mL n-butanol were added to extract lipids. The organic phase was obtained and fluorescence was determined at 515/553 nm excitation/emission (Modulus Microplate, Promega Corporation), using tetraethoxypropane as a calibrating standard.