Matrigel matrix high concentration hc

Hanging drop method was applied for cellular spheroid formation which has been described in our previous study [10 (link)]. Approximately 500 PC-3 and DU145 cells were added in 30 mL of solution, which contained Corning Matrigel Matrix High Concentration (HC) (Phenol-Red-free) and complete culture medium. VA (treatment group) or an equal amount of ddH₂O (NC group) was added into the liquid drops after 24-h incubation. The imaging was taken at 0 h, 48 h, and 96 h of drug treatment. The cross-section area was used to determine the 3D volume, which was calculated by ImageJ (version 1.52a; National Institutes of Health, USA). The cross-section area inhibition rate = (1-cross-section area of treated sample/cross-section area control sample [NC]) × 100%.

3-[4,5-Dimethylthiaoly]-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich) assay was performed to detect proliferation of SNs that were uninfected or infected by HIV-1 or ZIKV. 96-well flat bottom plates were pre-coated with Corning^® Matrigel^® Matrix High Concentration (HC), Growth Factor Reduced (GFR) ^∗LDEV-free treated according to the manufacturer’s recommendations. Differentiated iPSC-derived SNs were plated at 7 × 10⁴ per well of 96-well plates in 100 μL NS5B maturation medium, and were infected by HIV-1 or ZIKV in triplicate at MOIs of 0, 0.25, 0.5, or 1 for incubation (37°C, 5% CO₂) for 2 h, prior to replacing with fresh NS5B maturation medium and incubating (37°C, 5% CO₂) for another 24 h. The MTT assay was then carried out as recommended by the manufacturer, where media was replaced with freshly diluted MTT in PBS for a final concentration of 0.5 mg/mL, and plates were incubated (37°C, 5% CO₂) for 4 h prior to addition of the Solubilization solution, Plates were incubated (37°C, 5% CO₂), and absorbance was measured at 590 nm by a microplate reader (Bio-Rad).