For LSK lentiviral infection and rescue BMT, sorted LSK cells from either Hectd1f/f or Hectd1f/f;Vav (CD45.2) mice were cultured in SFEM media (StemCell Technologies Inc) supplemented with 10% FBS (SAFC Biosciences) and cytokines (100 ng/mL mSCF, 20 ng/mL mTpo, 20 ng/mL FLT3L, 20 ng/mL IL6) for 2 days. Lentivirus carrying mCherry/shLuc or mCherry/shmZNF622 were preloaded twice into a RetroNectin (T100B, Takara)-coated 12-well plate (Modlich et al., 2009 (link)). Cultured LSKs were transferred to the lentivirus-preload plates and incubated for one more day. At day3, 250k cultured LSKs were mixed with 500k Sca1-depleted competitor BM cells and injected into lethally-irradiated recipient mice. A small fraction of infected cells was spared for flow cytometry to evaluate the viral infection efficiency (Jiang et al., 2012 (link)).
Retronectin t100b
Manufactured by Takara Bio
RetroNectin (T100B) is a recombinant human fibronectin fragment that promotes cell adhesion and transduction efficiency in cell culture applications. It is a laboratory tool used to enhance the performance of cell-based assays and experiments.
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3 protocols using retronectin t100b
1
Lentiviral Infection and Rescue BMT
2
Lentiviral Transduction of Cell Lines
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293T cells were transfected with packaging plasmids and non-targeted lentivirus vector, or a lentivirus vector encoding shRNA targeting Mdm2 and c-Cbl. The virus containing supernatant was harvested 72 hours after transfection. LS 174T cells and Jurkat T cells were transfected with the lentivirus for 96 hours. Then, the cell lysates were analyzed by immunoblotting.
For overexpression, the cells were transfected with indicated plasmids by Lipofectamine 2000 reagent (Invitrogen) according to the instructions. 48 hours after transfection, the cell lysates were analyzed by immunoblotting.
For knockdown, the 6FAM labeled siRNA transfection method was used. Activated T cells were transfected in Nucleofector device using mouse T cell Nucleofector kit (VPA-1006, Lonza) according to the manufacturer instructions. Transfected cells were enriched and sorted based on 6FAM fluorescence (FACS Aria, BD Biosciences). For retronectin-mediated transduction, the virus-containing supernatants were loaded by centrifugation (2000 g, 2 hours at 32°C) onto 12-well plates pre-coated with RetroNectin (T100B, Takara). Activated T cells were transduced by centrifugation at 1000 g for 30 minutes at 32°C. Transfection was repeated after 24-hours incubation at 37°C in a humidified atmosphere containing 5% CO2. Transfected GFP+ T cells were sorted by flow cytometry sorter (FACS Aria, BD Biosciences).
For overexpression, the cells were transfected with indicated plasmids by Lipofectamine 2000 reagent (Invitrogen) according to the instructions. 48 hours after transfection, the cell lysates were analyzed by immunoblotting.
For knockdown, the 6FAM labeled siRNA transfection method was used. Activated T cells were transfected in Nucleofector device using mouse T cell Nucleofector kit (VPA-1006, Lonza) according to the manufacturer instructions. Transfected cells were enriched and sorted based on 6FAM fluorescence (FACS Aria, BD Biosciences). For retronectin-mediated transduction, the virus-containing supernatants were loaded by centrifugation (2000 g, 2 hours at 32°C) onto 12-well plates pre-coated with RetroNectin (T100B, Takara). Activated T cells were transduced by centrifugation at 1000 g for 30 minutes at 32°C. Transfection was repeated after 24-hours incubation at 37°C in a humidified atmosphere containing 5% CO2. Transfected GFP+ T cells were sorted by flow cytometry sorter (FACS Aria, BD Biosciences).
3
Lentiviral Transduction of Cell Lines
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