Retroviruses were produced in Plat-E cells. Plat-E cells were transfected with pMX-IRES-GFP plasmids containing the indicated genes, and the medium was replaced twice with 3 ml of fresh medium every 10 hours after transfection. The retrovirus-containing supernatant was collected 72 hours after the medium was replaced for the second time and used to infect T cells.

Sorted naive CD4+ T cells were differentiated into TH17 cells in the presence of TGF-β1 and IL-6 (48-well plate, 0.5 × 106 cells per well); 20 to 24 hours later, the cells were transfected with 1 ml of the indicated retrovirus in the presence of polybrene (10 μg/ml) and 10 mM Hepes and infected for 2 hours at 1500g at 32°C. After transfection, the cells were resuspended in TH17 differentiation medium and cultured for 3 days. The indicated cytokines (e.g., IL-17A and IL-17F) and other TFs (e.g., Foxp3 and RORγt) were measured by gated CD4+GFP+ cells after retrovirus infection for 72 hours.

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.