I am trying to phagocytc activity mesurament and the first step is mixing hemolymph with the Graces insect medium. But when I continued to collect hemolymph, it started to turn dark grey and black. Why is that?
The hemolymph is likely undergoing *melanization* due to:
1. *PO activation*: Prophenoloxidase (PO) enzyme activation, leading to melanin production.
2. *Oxidative stress*: Cellular damage or contamination triggering the melanization cascade.
To prevent this, use *anticoagulants* (e.g., EDTA, PTU) or collect hemolymph *on ice* to slow down enzymatic reactions.
The hemolymph turns dark because of melanization, a natural immune response in insects. Exposure to air or foreign surfaces activates phenoloxidase, which converts tyrosine into melanin, causing the hemolymph to darken. To slow this, keep hemolymph cold, minimize air exposure, and work quickly.
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