Luciferin substrate

The mouse cervico-vaginal model experiment was performed as described previously [57 (link)] with minor modifications (animal permit number G-93/17). The passive transfer assay was performed over the course of 8 days. On day 1, BALB/c male mouse cage bedding was transferred to the cages of female mice to induce hormonal synchronization (Whitten effect). On day 3, 100 μl of 30 mg/ml medroxyprogesterone acetate (Depo-Provera; Pharmacia) was injected subcutaneously. One hundred microliters of serum from immunized mice (diluted 1:1 with PBS) was delivered i.p. to each mouse on day 5. On day 6, mice were treated intravaginally with 50 μl of 4% Nonoxynol-9 (N9; Spectrum) in 4% carboxymethyl cellulose (Sigma). Four hours after N9 treatment, HPV pseudovirions encapsidating a firefly luciferase plasmid in 4% carboxymethyl cellulose (Sigma) were instilled intravaginally. Luminescence-based imaging using a Xenogen in vivo imaging system (IVIS) imager (Xenogen Corporation; PerkinElmer) was performed on day 8, and the efficiency of L2-directed immune responses was assessed after intravaginal instillation of 20 μl luciferin substrate (15 mg/ml; Promega). A region-of-interest (ROI) analysis was performed using Living Image 2.50.1 software (Xenogen; PerkinElmer). Background signal was obtained and subtracted by imaging each group of mice prior to instillation of luciferin.

Neutralization of replication-competent SHIVs was evaluated in vitro using TZM-bl target cells and a luciferase reporter assay as described [54 (link)–56 (link)]. Briefly, the SHIVs were generated by transfection in 293T cells. Viruses were incubated with antibody or plasma for 30 min at 37°C before TZM-bl cells were added. The HIV protease inhibitor indinavir was added to wells at a final concentration of 1 μM to limit infection of target cells to a single round of viral replication. Luciferase expression was quantified 48 hr after infection upon cell lysis and the addition of luciferin substrate (Promega).

The 293T human kidney cell line (HEK293T), and the C2C12 mouse myoblast cell line were cultured at 37°C in a humidified atmosphere of 5% CO₂ using DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco, USA). The cultures were split every 2 to 3 d. Lipofectamine 2000 (Invitrogen, USA) was used for the DNA transfections according to the manufacturer’s protocol.
C2C12 cells grown to 80% confluence in a 48-well plate were transfected with 100ng/well of the reporter construct along with 100 ng/well of pRL-TK plasmid (internal control) in the absence or presence of PfMSX expression vectors. At 48 h after transfection, the cultures were harvested and lysed. Luciferase assays were performed using 20 µl of cell extract and 100 µl of luciferin substrate (Promega, USA) using a luminometer.

HEK293T cells (1.5 × 10⁵ cells/well) were seeded onto 48-well plates. Cells were transfected with the pGL3 reporter gene in the absence or presence of PfBMP2 expression vectors. The total amount of DNA (1.0 μg) was kept constant with empty vectors. For normalization of transfection efficiencies, 0.1 μg of Renilla (sea pansy) luciferase expression plasmid (pRL-TK, Promega) was included in the transfection experiments. Transfected cells were lysed and subjected to luciferase assays using luciferin substrate (Promega) following the manufacturer's instructions. The assays were performed in triplicates.