FIG. 9.

Extracellular glutamine (Q) is required for the DN-to-DP transition promoted by ascorbic acid. OP9-DL1 cocultures were established and maintained without ascorbic acid for 19 days, at which time cultures were passaged into the conditions as indicated. The cell-permeable dimethyl-2-OG (DM2OG) was added at 1 mM, while ascorbic acid (AA) was added at 100 μM. Cultures were harvested 8 days later, and the total number of lymphoid cells per culture was determined using an Accuri C6 instrument. Cells were then labeled with antibodies for analysis by flow cytometry using a B-D Canto instrument. The absolute number of TCRαβ+ DN and DP cells in triplicate cultures was calculated and is shown as mean value±standard error.