Establishing Stable Lentiviral Cell Lines

For adherent Bel7402 cells, the cell number was diluted to 1.2×10⁵ cells/mL, and the cells were seeded into a 24-well plate at 500 μL/well. The culture was continued, and viral infection was performed when the degree of cell fusion reached 40%. For suspended THP-1 cells, The number of cells was diluted to 1×10⁵/mL, and 500 μL/well was seeded into a 24-well plate for direct viral infection. Then, 250 μL of fresh medium containing 1×Hitans GP or 1×Hitans GA was added, and the corresponding virus volume was converted according to the selected multiplicity of infection(MOI) gradient (10 (link), 24 (link)–26 (link)) and added to fresh medium containing the viral infection booster solution. Cell culture plates were shaken using the crossing method. After 4 h, 250 μL of fresh medium containing the infection booster solution was added again for 15 h. The cells were washed twice with sterile PBS, and fresh virus-free medium was added. The efficiency of the viral infection was observed after 48 and 72 h. After the puromycin concentration was screened, cells in the blank group were seeded in a 24-well plate, and the culture medium was replaced with fresh medium containing puromycin after 24 h. The puromycin screening gradients was 0.6, 1.2, 1.8, 2.4, and 3.0 μg/mL. The fresh medium was replaced according to the cell state, and the minimum puromycin concentration that killed all cells in the blank group for 3-4 days was selected as the experimental concentration of infected lentiviral cell lines. To screen stable virus-infected cell lines, 72 h after lentiviral infection, the concentration of puromycin found in the pre-experiment was used to simultaneously screen the lentivirus-infected and blank groups. After the cells in the blank group died completely, the concentration of puromycin in the lentivirus-infected cells was reduced to 50% of the original concentration and the culture was maintained. After 3 days, the medium was replaced with fresh medium without puromycin, and the obtained cells were considered THP-1 stable cells.