Subcellular fractionation of human neutrophils on Percoll density gradients.
Subcellular fractionation has been an important tool in the investigation of neutrophil structural organization including granule heterogeneity, composition and mobilization. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nitrogen cavitation as an efficient but gentle means of disrupting neutrophils and with Percoll as a density medium. This paper describes in detail the methodology of subcellular fractionation of nitrogen cavitated neutrophils on one-, two-, and three-layer Percoll density gradients. Appropriate marker proteins are presented for neutrophil organelles including azurophil, specific and gelatinase granules, in addition to secretory vesicles and plasma membranes. The dynamics of granule and secretory vesicle exocytosis is demonstrated by subcellular fractionation of resting and activated human neutrophils. Finally, the paper describes the applications of subcellular fractionation in the investigation of the localization of neutrophil constituents, in protein purification schemes and in the study of translocation of cytosolic proteins to isolated neutrophil organelles.
Kjeldsen
L
L
Granulocyte Research Laboratory, Department of Hematology, The Finsen Center, The National University Hospital, Rigshospitalet, 9 Blegdamsvej, 2100, Copenhagen, Denmark. lkjeld@rh.dk
Sengelov
H
H
Borregaard
N
N
D001692:Biological Transport; D015415:Biomarkers / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods*; D003102:Colloids; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D018375:Neutrophil Activation; D009504:Neutrophils / Q000737:chemistry* / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D009584:Nitrogen; D011205:Povidone; D011506:Proteins / Q000302:isolation & purification; D012822:Silicon Dioxide; D013347:Subcellular Fractions / Q000737:chemistry; D013395:Sucrose
D015415:Biomarkers; D003102:Colloids; D011506:Proteins; D013395:Sucrose; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; D009584:Nitrogen