I have isolated Naïve CD4 T cells from mouse spleen and tried to polarize them into Th1 cells. For doing that, I have coated a 48-well plate with 1 µg/ml Anti-CD3, 100 µl each well, incubated at 4℃ overnight. Then, I discarded the remaining anti-CD3 and didn't washed the wells with PBS. Then. I seeded 5*10^5 cells/500 µl complete RPMI. I divided cells into two populations to see the differences in polarization; 1. Th0 cells, receiving anti-CD28 (2µg/ml), and 2. Th1 cells, receiving Anti-CD28 (2µg/ml), human-IL-2 (150 IU/ml), IL-12 (10ng/ml), and anti-IL-4 (5µg/ml). Cultured the cells for three days and on day 2, I added fresh medium containing the same set of cytokines. Then I evaluated the expression level of T-bet using flowcytometry and I found that there is no difference between the two populations! Both Th0 and Th1 cells expressed T-bet around 70%, which is strange! I was wondering what is the reason? Can anti-CD3 and anti-CD28 promote the expression of T-bet similar to Th1 ?! Or was the stimulation so strong? I'd appreciate if someone can share a Th1 polarization protocol so I can check mine.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining