Mast Cell Degranulation Assay to Study the Effect of a Target Chemical

Mast cells — specialized white blood cells, contain secretory granules rich in lysosomal enzymes, such as beta-hexosaminidase.

To perform mast cell degranulation — a process involving the release of secretory granules, begin with a multi-well plate containing a murine peritoneal mast cell suspension pre-treated with immunoglobulins.

Treat the cells with a target chemical. The target chemical with stimulating potential interacts with pre-bound immunoglobulins on mast cells' specific receptors. This activates the movement of granules containing beta-hexosaminidase to the cell surface, releasing it into the solution.

Centrifuge the plate to pellet the cells, and transfer the enzyme-containing supernatant into an empty well of a fresh plate. Treat the cell pellets with a lysis buffer to lyse the cells and release their contents into the lysate, including intracellular beta-hexosaminidase.

Add the enzyme-containing lysate into another well of the same multi-well plate. Treat both wells with the enzyme's substrate, p-nitrophenyl-acetyl-D-glucosamine, and incubate. The enzymes hydrolyze the substrates, releasing p-nitrophenols — a colored product that makes the solution yellow.

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