Example 4
A fourth generation lentivirus vector system was used. PD1/CD28 vector, packaging vector pMDL-gag, Rev, and envelop vector pMD2.G were co-transfected into HEK293T cells with calcium phosphate or liposome-PEI. The supernatant was collected after 48 hrs, and centrifuged to concentrate the lentivirus.
Lentivirus titration was conducted on a three-fold serial dilution. HEK293T cells were collected after transduction with 50 ul lentivirus for 48 to 72 hrs, and then stained with PD-1. The percentage of PD-1+(PD-1+%) was analyzed by flow cytometry, and titration was calculated as:
Titration (TU/ml)=40000-45000(which is the number of starting HEK293T cells)*PD1+%*Fold of dilution*20 (first PD1+%<20%)
