293T, HCT-116, and U2OS were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Gaithersburg, MD) containing 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Gibco) in a CO2 incubator (5% CO2) with normal atmospheric oxygen tension (~21% O2). mESCs were grown in 2i medium as described in a previous study (16). Human primary endometrial stromal cells (hEnSCs) were derived and maintained in culture as previously described (9). Low-passage (up to 12) hEnSCs were used to perform the experiments described in this study. Transfections and viral transduction were performed using previously described methods (30). Specifically for hEnSC viral transduction, we used concentrated virus stocks. These were generated by ultracentrifugation at 25,000 rpm for 2.5 hours of supernatant containing virus using 32.4-ml OptiSeal tubes (Beckman Coulter) with the SW 32 Ti rotor (Beckman Coulter) and the Optima XPN-100 ultracentrifuge (Beckman Coulter). In this case, 2 × 106 hEnSCs were infected by adding lentivirus particles coming from the equivalent of two 10-cm dish (approximately 20 ml) supernatants resuspended in 10 to 20 μl of DMEM in hEnSC-specific medium (9). Polybrene was added as previously described (30). The multiplicity of infection for hEnSC used was ~1. All the cells transduced with lentivirus were positively selected using the appropriate antibiotic according to the resistance expressed by the lentiviral vector [puromycin (2 μg/ml), hygromycin (300 to 500 μg/ml), and neomycin (500 to 900 μg/ml)] until untransduced control cells treated with antibiotic were cleared from the plate. All the cells presented expressing exogenous proteins in this study were stably transduced unless indicated in the figure legend.

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

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.