Institute for Medicine and Engineering, University of Pennsylvania
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Oh, H., Siano, B., Diamond, S. Neutrophil Isolation Protocol. J. Vis. Exp. (17), e745, doi:10.3791/745 (2008).
Neutrophil Isolation Protocol
The density gradient separation method is used to isolate human neutrophils from whole blood using a mixture of sodium metrizoate and Dextran 500. This method is based on the mononuclear leukocyte separation method by Boyum (1968) which was modified for neutrophil separation by Ferrante and Thong (1980).
After collection from a donor, whole blood may be anticoagulated with EDTA, citrate, or heparin. Since they are short-lived, neutrophils should be used within 2-4 hours of collection. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended to desired concentration.
If the six bands are not distinct after the first centrifugation step, the separation process was not clean and will need to be repeated. For clean separation, make sure that the isolation media has not expired or contaminated. Separation may also be unsuccessful if the blood donor has consumed alcohol or medications in the 72 hours prior to blood collection.
To prevent neutrophil activation during the separation procedure, it is best to use HBSS without Ca2+/Mg2+, since the ions have been shown to prime cells. Resuspension of pellets should also be performed slowly, and vortex settings should be kept in the low- to mid-range so that cells do not become activated.
When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability. Purity and viability can be assessed by labeling the cells with neutrophil-specific marker CD66b and trypan blue dye exclusion.
Funding from NIH R01 HL56621.
|Lymphocyte Poly(R)||Reagent||Cedarlane Labs||PolymorphprepTM can be used as an alternative|
|Hank’s Balanced Salt Solution without Calcium Chloride||Reagent||Invitrogen|
|Human Serum Albumin||Reagent||ZLB Bioplasma|
|Red Cell Lysis Buffer||Reagent||Roche Group|
|Pasteur Pipettes and bulb||Tool|
|PolymorphprepTM||Reagent||Axis-Shield||Alternative reagent to Lymphocyte-Poly (R)|
|Syringe Filter||Other||EMD Millipore||SLGV033RS||Millex-GV, 0.22 Âμm, PVDF, 33 mm, gamma-sterilizable|
|Hank’s Balance Salt Solution with Calcium Chloride||Reagent||Invitrogen|
Boyum, A. Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Invest. 21, Suppl. 97, 77-83 (1968).
England, J.M., Rowan, R.M. et al. The assignment of values to fresh blood used for calibrating automated blood cell counters. Clin. Lab. Haemat. 10, 203-212 (1988).
Garcia, A. et al. Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay. J. Clin. Microbiol. 34, 182-184 (1996)
Ferrante, A. & Thong, Y.H. Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the Ficoll-Hypaque method. J. Immunol. Methods. 36, 109 (1980).