After isolating neutrophils from human peripheral blood, resuspend them in RPMI 1640 Medium in a 1.5 milliliter centrifuge tube. Add one microliter of membrane permeable red DNA die to 1.5 milliliters of neutrophil suspension and incubate in the dark for five minutes. Centrifuge the neutrophil suspension at 2, 500g for five minutes at room temperature.
After centrifugation, remove the tube containing pelleted neutrophils and aspirate the supernatant. Resuspend the neutrophils in one milliliter of RPMI and centrifuge it again. After the last wash, resuspend the neutrophils in one milliliter of RPMI.
Add a small volume of neutrophil suspension to the hemocytometer and count them under a microscope. Dilute the neutrophil suspension to 1.5 times 10 to the fifth neutrophils per milliliter using RPMI. Add four microliters of one to 100 pre-diluted membrane impermeable green DNA dye to the one milliliter of neutrophil suspension.
Dispense 100 microliters of neutrophil suspension per well into a clear tissue culture treated 96 well plate. Add 100 microliters of RPMI containing stimulus reagents with or without inhibitor into the respective wells. Place the plate in a live cell analysis system housed in a 5%carbon dioxide incubator.
