Isolating primary monocytes from the C57BL/6 femur and Inducing differentiation BMDC cells by adding 10 𝛍g/ml GM-CSF and 10 𝛍g/ml IL-4 to the mediu. Replace with 75% fresh medium on day 2, replace with 100% fresh medium on days 3 and 5, resuspend cells and add samples to stimulate cell activation on day 6. FACS was evaluated by detecting CD11c, CD80, and CD86. Curiously, after the first successful activation, the BMDC could not be activated after the second replacement of the added cytokines, and the same sample was used at the same concentration.The difference is that the BMDC differentiates rapidly under the second neocytokine induction, and the previous large number of clusters began on day 4-5, and the second time began to cluster a lot on day 3.What is the possible reason for this? I also charged the cells on day 6 with samples stimulated for 12 h to take QRT.The mRNA expression of IFN-𝛼 and IFN-𝛃 In the case of the first large number of expressions, there was no significant difference between the second and the blank control.I would be grateful if any of my classmates or researchers could answer any questions I encountered.