The pMSCV Exp-mCcr4 NM_009916.2 (ns): P2A: EGFP (Vector ID: VB230314-1872tcs) vector was designed and created using VectorBuilder, Inc. (Guangzhou, China). The vector was then packaged into a recombinant MSCV retrovirus using VectorBuilder, Inc. (Guangzhou, China).
The general procedure for Treg cells was transduced as previously described24 (link). Transduction media was prepared with RPMI media with 100 mM HEPES and 10 μg/mL polybrene. We carefully pipetted 100 μL of the virus stock into each well containing 1 mL of the transduction media. The culture was spinoculated at 1,200 g for 90 min at 32 °C. Cells were incubated under standard culture conditions for 4 h, followed by a media change. Treg cells were sorted on days 5 or 6 using a BD Aria III (Becton Dickinson Company, Franklin Lakes, New Jersey).
The general procedure for Treg cells was transduced as previously described24 (link). Transduction media was prepared with RPMI media with 100 mM HEPES and 10 μg/mL polybrene. We carefully pipetted 100 μL of the virus stock into each well containing 1 mL of the transduction media. The culture was spinoculated at 1,200 g for 90 min at 32 °C. Cells were incubated under standard culture conditions for 4 h, followed by a media change. Treg cells were sorted on days 5 or 6 using a BD Aria III (Becton Dickinson Company, Franklin Lakes, New Jersey).
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