Luciferase assay system

The ADE assays were performed using Raji cells. A total of 50 µL of serially diluted mAbs or mAbs combinations were mixed with 50 µL of supernatant containing 750 TCID₅₀ of pseudovirus. The mixture was incubated for 60 min at 37 °C, and then supplied with 5% CO₂. A total of 100 µL cells at a density of 2 × 10⁶ cells/mL were added to the mixtures of pseudoviruses and mAbs for an additional 24 h incubation. Then, the same volume of luciferase-detecting regents (Beyotime) was added to each well. After 2 min of incubation, the luciferase activity was measured by the Luciferase Assay System (Beyotime). The maximum ADE infectivity fold-change was calculated by comparing the peak ADE luciferase levels to those in the virus control wells (containing the virus and cells). This calculation was made after subtracting the background levels observed in the control groups containing cells only.

LNCaP and PC3 cell were plated on 12-well plates and transfected using Lipofectamine 2000 (3 mL per well; Invitrogen). The total amount of plasmid DNA was normalized to 1.5 μg per well. After 48 hr, cell were lysed in lysis buffer provided in the Luciferase Assay System (Beyotime Biotechnology). Luciferase activities were measured using the Dual-luciferase Reporter Assay System (Beyotime Biotechnology) with the aid of a microplate luminometer (Tecan, Switzerland), and Renilla luciferase activities of cell were used as internal control. All experiments were carried out in triplicate wells and repeated 3 times.

In order to perform neutralization experiments, pseudoviruses were incubated with serial dilutions of sera, and the reduction in luciferase gene expression was determined. Briefly, 2 × 10⁴ Vero E6 cells were seeded per well in a 96-well plate. On the next day, the pseudoviruses were incubated with triple serial dilutions of the test samples for 30 min at 37 °C. Then, the cells were added into the mixture and cultivated for another 24 h. The Luciferase Assay System (Beyotime, Shanghai, China) was used to measure the luminescence. The half-maximum concentration (IC₅₀) was defined as the dilution for which the relative light units were reduced by 50% compared with the virus control (virus + cells) after subtracting the background of the blank control groups (cells only). The IC₅₀ values were calculated using nonlinear regression in GraphPad Prism. The half-maximal inhibitory concentration (IC₅₀) represents the dilution factor that reduces the relative light unit by 50% compared to the virus control (virus + cell) after subtracting the background of the blank control group (only cells). To calculate this parameter, we used the scientific computing software GraphPad Prism v.10 to calculate the IC₅₀ value through nonlinear regression.

HIV-1 latent infection was done following a previous report^{61 (link)}. Briefly, PBMCs were isolated using Ficoll density gradient centrifugation (GE Life) from buffy coats of two healthy anonymous donors. Naïve CD4 + T cells were isolated by a naïve human CD4 + T-cell enrichment kit (Miltenyi Biotec). CD4 + T cells were cultured with CD3/28-Dynabeads for 3 days; the Dynabeads were then removed and cultured with IL-2 (30 IU/ml) for 4 days. CD4 + T cells were infected with NL4-3/Luc pseudovirus and were then washed with prewarmed RPMI with IL-2 and plated on a 12-well plate the next day. At 7 dpi, cells were transduced with shRNA-scramble or shRNA-TRIM5α lentiviruses, followed by 500 ng/ml puromycin treatment at 10 dpi. A portion of the cells was used for Western blotting to check the knock-down efficiency of shTRIM5α at 13 dpi before cells were reactivated using CD3/28-Dynabeads. Three days after reactivation, cells were washed with PBS and lysed by 100 μl passive lysis buffer for luciferase assay. The luciferase activity in cell extracts was quantified by mixing 50 μl of lysate with 50 μl of substrate (Luciferase Assay System, Beyotime).

Neutralization assays were performed by incubating pseudoviruses with serial dilutions of monoclonal antibodies or sera, and scored by the reduction in luciferase gene expression. In brief, Vero E6 cells were seeded in a 96-well plate at a concentration of 2×10⁴ cells per well. Pseudoviruses were incubated the next day with serial dilutions of the test samples in triplicate for 30 min at 37 °C. The mixture was added to cultured cells and incubated for an additional 24 h. The luminescence was measured by Luciferase Assay System (Beyotime). IC₅₀ was defined as the dilution at which the relative light units were reduced by 50% compared with the virus control wells (virus + cells) after subtraction of the background in the control groups with cells only. The IC₅₀ values were calculated using nonlinear regression in GraphPad Prism.

Neutralization assays were performed by incubating pseudoviruses with serial dilutions of monoclonal antibodies or sera, and scored by the reduction in luciferase gene expression as described previously^{51 (link)}. In brief, Vero-E6 cells were seeded in a 96-well plate at a concentration of 2 × 10⁴ cells per well. On the following day, pseudoviruses were incubated with serial dilutions of the test samples in triplicate for 30 min at 37 °C. The mixture was added to cultured cells and incubated for an additional 24 h. The luminescence was measured by the Luciferase Assay System (Beyotime). IC₅₀ was defined as the dilution at which the relative light units were reduced by 50% compared with the virus control wells (virus + cells) after subtraction of the background in the control groups with cells only. The IC₅₀ values were calculated using nonlinear regression in GraphPad Prism.

To detect luciferase activity in engrafted AD-MSCs, heart tissues were removed from sacrificed mice on POD14, homogenized in PBS containing a protease inhibitor cocktail (Selleck, USA), and lysed with 1×PLB(passive lysis buffer). Luciferase activity in the supernatant was measured using the Luciferase Assay System (Beyotime, China) on GloMaxTM 20/20 Luminometer (Promega, USA) following the protocols we previously described [22 (link)].