I am trying to make a primary culture of microglia from offspring, and I am having a big problem. The protocol I am using begins with the dissociation of the cortex with 0,05% of Trypsin for 10 minutes and then the mechanical dissociation with pipette (P1000). After that, the cells are passed through a 70µm membrane and plated in 75cm2 bottles, 1 cortex per bottle. The next day the medium is changed and the culture left in the incubator( 37°C 5% CO2). The problem that I have is that in the second day appear some small cells that prevents the microglia to grow. These cells are adherents and don’t seems to be bacterial or fungus because they don’t modify the medium. I repeated this protocol for at least 3 times and this continues to happen. Has anyone had this problem and can you help me to solve this?