This article details the process of mast cell degranulation, focusing on the release of beta-hexosaminidase from murine peritoneal mast cells. The methodology includes treating cells with target chemicals and measuring enzyme release spectrophotometrically.
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.
To measure beta-hexosaminidase release, transfer 100 microliters of the cell suspension per well to a 96-well V-bottom well plate. Add 25 microliters of either stimulation or control solution to each well. Then, incubate the cells for 45 minutes at 37 degrees Celsius. Afterward, stop the reaction by placing the 96-well plate on ice for 5 minutes. Then, centrifuge the plate at 4 degrees Celsius for 4 minutes at 120 g.
Transfer 120 microliters of the supernatant to a flat-bottom 96-well plate, and place it on ice. Carefully avoid touching the cell pellets, but completely aspirate the supernatant. Next, add 125 microliters of lysis buffer to the cell pellets. Incubate the cell pellets for 5 minutes at room temperature, and resuspend them after the incubation by repeated pipetting.
Pipette 25 microliters of the 4-millimolar pNAG solution in the required wells of a new flat-bottom 96-well plate. Add 25 microliters of each supernatant, and 25 microliters of each cell lysate separately to the prepared pNAG solution. Incubate the reaction batches for 1 hour at 37 degrees Celsius. After the incubation, pipette 150 microliters of stop solution to each well to stop the reaction.
Next, analyze the plate sample light absorbance with a microplate reader at 405 nanometers with reference 630 nanometers for automatic background subtraction. In the acquisition program, check the box, "Reference", and in the "Absorbance" program element menu, select "630 nanometers" for automatic background subtraction.
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