I performed a Percoll protocol (gradient 72%-62%), but I observed 2 rings, one on the surface and another between the gradients. Which is the ring that represents mature neutrophils?
The only absolute way to determine which band correlates with the mature neutrophils would be to separate both layers and run a flow panel on both layers.
As a side note, A far superior and cleaner way to isolate a pure neutrophil population is to use a magnetic bead separation assay. Preferably for this you could use a neutrophil enrichment kit which would deplete all other cells in the Bone marrow and leaving your neutrophils untouched. StemCell technologies sells a column free alternative to the Miltenyi magnetic columns. However, both work quite well and return a ~99-95% purity depending on methodology and kit.
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