LSK and LK sorted cells were stimulated overnight in StemSpan serum-free expansion medium (STEMCELL Technologies) supplemented with rmTPO (recombinant mouse thrombopoietin) (10 ng/ml; R&D Systems), rmFlt3L (50 ng/ml; R&D Systems), and rmSCF (100 ng/ml; R&D Systems). Hematopoietic progenitors were transduced using RetroNectin (Takara Bio Inc.)–coated wells according to the manufacturer’s instructions. Nontissue culture plates were coated with RetroNectin overnight at 4°C and then blocked with 2% BSA in PBS for 30 min. Retroviral supernatant (24 or 48 hours) was centrifuged at 1500g for 1 hour at 32°C and incubated an extra hour at 37°C. After coating, viral supernatant was removed and stimulated cells were immediately added on the virus-coated plates. Cells were cultured in StemSpan medium supplemented with rmTPO (10 ng/ml), rmFlt3L (50 ng/ml), and rmSCF (100 ng/ml) and transduced overnight at 37°C. LZRS-ires-eGFP–, LZRS-Gata3-ires-eGFP–, and LZRS-Bcl11b-ires-eGFP–transduced cells were used for in vitro and in vivo approaches.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.