Related Experiment Video
Updated: Jun 20, 2026
Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue
Published on: August 15, 2012
Microglia of rat ventral midbrain recovers its resting state over time in vitro: let microglia rest before work
Ana Clara Cristóvão1, Ana Saavedra, Carla Pais Fonseca
1Health Sciences Research Center, University of Beira Interior, Covilhã, Portugal.
Abstract:
Cortical or total brain cultures of microglia are commonly used as a model to study the inflammatory processes in Parkinson's disease. Here we characterize microglia cultures from rat ventral midbrain and evaluate their response to zymosan A. We used specific markers of microglia and evaluated the morphology, the phagocytic activity and reactive oxygen species (ROS) levels of the cells. During the first 10 days in vitro (DIV), cultures presented predominantly cells with a round morphology, expressing CD68 and with high phagocytic activity and ROS production. After 13 DIV, this tendency was reversed, with cultures showing higher number of ramified cells and fewer CD68(+) cells along with lower phagocytic and ROS production capability, suggesting that microglia must be kept in vitro for at least 13 days to recover its resting state. The exposure of cultures with less than 10 DIV to zymosan A significantly decreased cell viability. Exposure of cultures with 13 DIV to zymosan A (0.05, 0.5, or 5 microg/ml) increased the total cell number, the percentage of CD68(+) cells, and the phagocytic activity. Concentrations of zymosan A higher than 5 microg/ml were also effective in activating microglia but significantly decreased the number of viable cells. In summary, microglial cells remain in the activated state for several days after the isolation process and, thus, stimulation of microglia recently isolated can compromise interpretation of the results. However, upon 13 DIV, cells achieve properties of nonactivated microglia and present a characteristic response to a proinflammatory agent.
Insights
Microglia cultures from rat ventral midbrain are activated for days after isolation. For accurate Parkinson's disease research, cultures need at least 13 days in vitro to reach a resting state before stimulation.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are key immune cells in the brain, crucial for neuroinflammation research in diseases like Parkinson's.
- In vitro microglia cultures are widely used models, but their activation state post-isolation is not fully understood.
Purpose of the Study:
- To characterize primary microglia cultures from rat ventral midbrain.
- To evaluate the impact of in vitro culture duration on microglia activation state.
- To determine optimal conditions for using microglia cultures to study inflammatory responses.
Main Methods:
- Primary microglia cultures were established from rat ventral midbrain.
- Cell morphology, CD68 expression, phagocytic activity, and reactive oxygen species (ROS) levels were assessed over time in vitro (DIV).
- Response to zymosan A stimulation was evaluated at different time points (before and after 13 DIV).
Main Results:
- Microglia cultures exhibited an activated phenotype (round morphology, high CD68, high phagocytosis, high ROS) for the first 10 DIV.
- After 13 DIV, cultures shifted towards a resting phenotype (ramified morphology, low CD68, low phagocytosis, low ROS).
- Zymosan A stimulation of cultures <10 DIV reduced cell viability, while stimulation of cultures ≥13 DIV enhanced cell number and activation markers without significant toxicity at optimal concentrations.
Conclusions:
- Newly isolated microglia cultures remain activated for several days, potentially confounding experimental results.
- At least 13 DIV in vitro is required for microglia to regain a resting state and exhibit a characteristic response to inflammatory stimuli.
- These findings are critical for optimizing experimental design in neuroinflammation and Parkinson's disease research using microglia models.
