LAD2 Cell Degranulation Assay

LAD2 cells were grown in StemPro-34 medium (Gibco) supplemented with 100 μg/mL SCF (Novoprotein), 100 μg/mL IL-6 (Novoprotein), nutrient supplement (NS) (Gibco), 100 U/mL penicillin (Invitrogen), 100 μg/mL of streptomycin (Invitrogen) and 2 mM L-Glutamine (Gibco) at 37°C under 5% CO₂. LAD2 cell degranulation was evaluated by measuring the release of β-hexosaminidase (Hsieh et al., 2019 (link)). Briefly, LAD2 cells (3.5 × 10⁵) were exposed to HIV-JRFL/VLP (10 or 100 ng p24^Gag) or HIV-HXB2/VLP (10 or 100 ng p24^Gag) for the indicated times. C48/80 (4 μg/mL) was used to induce MC degranulation as the positive control. For measuring β-hexosaminidase activity, the substrate of p-nitrophenyl-N-acetyl-β-D-glucosaminide was dissolved in 0.1 M sodium citrate (pH 4.5) for reaction for 1 h at 37°C, then 0.1 M carbonate buffer (pH 10) was added to stop the reaction. The product of 4-p-nitrophenyl was detected at absorbance of 405 nm. β-hexosaminidase was measured in the supernatant as well as the cell lysate solubilized in 0.1% Triton-X100. Percentage of degranulation was calculated by dividing the absorbance in supernatant by the sum of absorbance in both supernatant and cell lysate.