4.6. Estimation of Lentiviral Titer by alamarBlue Assay

The transduction unit (TU) of the SARS-CoV-2-pseudotyped lentivirus was estimated via cell viability assay in response to the limited dilution of the lentivirus. In brief, HEK-293T cells stably expressing the human ACE2 gene were plated on 96-well plates one day before lentivirus transduction. For the titering of pseudotyped lentivirus, different amounts of lentivirus were added into the culture medium containing polybrene (final concentration 8 µg/mL). Spin infection was carried out at 1100× g in 96-well plates for 30 min at 37 °C. After incubating cells at 37 °C for 16 h, the culture medium containing virus and polybrene were removed and replaced with complete DMEM containing 2.5 µg/mL puromycin. After treating puromycin for 48 h, the culture medium was removed and the cell viability was detected by using 10% alamarBlue reagents, according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The survival rate of uninfected cells (without puromycin treatment) was set as 100%. The virus titer (transduction units; TU) was determined by plotting the surviving cells versus the diluted viral dose [45].