Mast Cell Degranulation Assay to Study the Effect of a Target Chemical
Trascrizione
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.
Overlay the well with an alkaline-stopping solution to terminate the reaction and convert p-nitrophenol to p-nitrophenolate, intensifying the yellow coloration. Spectrophotometrically estimate the solution's absorbance for both wells at 405 nanometers.
Calculate the percentage release of enzymes that correlates with the stimulating effect of the target chemicals on mast cell degranulation.
