NitrogenBomb1)Harvestcellsandwash2timeswithPBS.2)ResUSPendfinalpelletinrelaxationbuffer.(20ml/1X109cells)3)PressurizewithN2for20minutesat350psi,40Cwithconstantstirringinanitrogenbomb.4)CollectcavitatedropwiseintoEGTA,pH7.4.-->ThefinalEGTAconcentrationshouldbe1.25mM.Fractionation5)Pelletthenucleiandunbrokencellsbycentrifugingthecavitateat500g,40Cfor10min.-->ThispelletisknownastheP1fraction.6)Thesupernatantshouldbedecantedandlayeredontoaprecooled(to40C)PercollgrADIent. NOTERelaxationbuffer,ahigh-potassium,low-sodium,calcium-freebuffercontainingMgATP,wasdesignedtomimiccytoplasmicconditionsintheneutrophil,basedinpartuponconditionsshowntopromotecytoplasmiocrelaxationinnonmusclecontractilesystems.
ReagentsRelaxationbuffer(withoutEGTA):100mMKCl3mMNaCl1mMATP(Na)23.5mMMgCl210mMPIPESpH7.3
ReferenceBorregaard,N.etal.(1983)J.CellBiol.97,52-61.