I’m culturing primary mouse embryonic motor neurons and encountering issues with neuronal health and clustering, especially around DIV 3–4. I’ve attached a representative image from DIV 3 showing what appears to be excessive clustering and significant cell debris (black granules). The neurons don’t appear healthy, and similar conditions are seen across both infected and non-infected wells. (i have tried seeding on Cover slips or without and it didnt help !

Some key observations:

• Clustering: Neurons start forming dense clusters early (around DIV 3), which often correlates with poor viability and reduced neurite outgrowth.
• Cell Debris: There’s substantial granular debris visible, suggesting cell death, but there are no signs of microbial contamination.
• Health Status: Even neurons in control wells (not exposed to virus) are unhealthy, suggesting the problem is not due to viral toxicity.
• I use Neurobasal + B27 + GlutaMAX, with BDNF and GDNF.
• Media is pre-warmed at 37°C in a 5% CO₂ incubator for 20–30 minutes before use
• Plates are coated with Poly-DL-Ornithine (PLO) (0.1 mg/mL) overnight at 37°C, then washed with UPW, followed by Laminin for 2 h at 37°C.

I’ve attached images taken at DIV 2 and DIV 5, showing how the cultures evolve. the culture seems good at first and then it just went all wrong and i dont understand why

i have this proplem very long time now , the clusters and a very bad looking and unhealthy neurons so i will so happy if someone can help me with that, each tip will be helpfull , thank you !