For my project, I have been trying to separate microglia from mixed culture using shaking method but have not yet been successful. Astrocytes manage to creep in after shaking and ultimately over populate in the next passage.
Panini Patankar 🧪 Overview of the Procedure
🧬 Detailed Protocol
1. Dissection and Preparation of Mixed Glial Cultures
- Use P0–P2 neonatal Sprague-Dawley rats.
- Dissect the cerebral cortex under sterile conditions.
- Remove meninges carefully.
- Mechanically dissociate (trituration using pipette) in ice-cold HBSS or DMEM/F12.
- Centrifuge and resuspend in complete glial medium (e.g., DMEM/F12 + 10% FBS + pen/strep).
- Seed the cells in T75 flasks (or similar) pre-coated with poly-D-lysine.
2. Culture Maintenance (10–14 days)
- Incubate at 37°C with 5% CO₂.
- Change medium every 3 days.
- Over time, a layer of astrocytes will form at the bottom, while microglia float on top or loosely attach.
3. Shaking to Isolate Microglia
- On Day 10–14: Tighten the flask cap. Shake the flask on an orbital shaker: 180–200 rpm for 1–2 hours at 37°C (shorter for enriched microglia). This step dislodges microglia, while astrocytes remain attached.
4. Collect and Purify Microglia
- Carefully collect the supernatant (contains microglia).
- Centrifuge at 300×g for 5–10 min to pellet microglia.
- Wash with PBS or fresh medium.
- Optionally, resuspend and plate microglia in: Uncoated plates (for floating microglia) Or poly-L-lysine coated plates (to allow adherence and further experiments)
🔬 Optional Enhancements
- Purity Check: Use Iba1 or CD11b immunostaining to confirm microglia identity.
- Microglia Activation Assays: After plating, you can treat cells with LPS or IFN-γ to study inflammation.
- Astrocyte Purification: If you want astrocytes, try a second shake at higher speed (240 rpm for 6 hours) to isolate oligodendrocyte precursor cells and leave behind pure astrocytes.
⚠️ Notes & Tips
- Microglia yield and viability can vary depending on: Age of pups (P2 is fine, P0 often yields more) Shaking time and intensity Culture confluency
- Avoid over-shaking, as it may damage astrocyte layer or detach unwanted cells.
Thanks a lot Vigneshwaran Subharayan I am exactly following the same protocol and I have been successful is separating astrocytes but with microglia I am always getting mixed culture.
Elisabeth Petrasch-Parwez I was not able to connect with him as I could not find him on research gate. I would be grateful if you can help me connect with him
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