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