I am using the OP9-DL1 co-culture system to differentiate mouse bone marrow cells into double positive T cells. I infected the bone marrow cells with a retrovirus, selected the GFP+ cells, and have co-cultured them for 35 days on OP9-DL1 cells with 5ng/ml FLT-3L and IL-7 (passaging every 3-4 days). They are arresting at the DN3/DN4 stage before becoming DP T cells. According to the literature IL7 could prevent differentiation so I co-cultured a subset of cells at Day 21 with 0ng/ml IL7 and 0.2ng/ml IL7 this greatly reduced cell number and pushed cells to be exclusively DN4 (Day 28) and DN4 with some CD44+ cells (Day 35) but still no DP T cells. They are still growing in culture. I was wondering if anyone has encountered this particular problem or has any advice.
May be your cells don't expression Notch1 in the surface. Because we know that Notch-1 has been shown to be critical for the establishment of T cell fate. Loss of function results in the blockade of T cell development.
- Badges
- Science topic