Human Peripheral blood's Neutrophil isolation

Sam Coward sam.coward at sunderland.ac.uk
Thu Aug 2 09:49:40 EST 2001


This is the method we use in our lab;

Neutrophil Isolation
Neutrophils were isolated from venous blood of healthy volunteers by dextran
sedimentation of erythrocytes and density-gradient centrifugation of
leukocytes. All
procedures up to and including density-gradient centrifugation were conducted
at 22° C unless stated otherwise. Blood was anticoagulated with
acid-citrate-dextrose
and centrifuged at 400 × g for 20 min to remove platelets. The platelet-rich
plasma supernatant was removed and centrifuged at 2,000 × g for 30 min to
prepare
platelet-poor plasma (PPP). The platelet-depleted cell precipitate from the
first centrifugation step was mixed with phosphate-buffered saline (PBS) (1:4).
Dextran
(Baxter, Warrington, UK) was added to a final concentration of 2% and
erythrocytes were sedimented under gravity for 45 min at 15° C. The
erythrocyte-depleted supernatant containing leukocytes was centrifuged at 400 ×
g for 10 min. The resulting leukocyte-enriched pellet was resuspended in PPP.
The leukocytes were then layered over a discontinuous gradient of Percoll (4 ml
of 61.5% and 4 ml of 76%) in a 15-ml polypropylene centrifugation tube. The
Percoll densities were created from pure Percoll diluted with 10× PBS and PPP.
Final percentages of PPP (vol/vol) in the Percoll solutions were 42.89% and
23.97% for the 61.5% Percoll layer and 76.0% layer, respectively. The Percoll
densities and layered cells were then centrifuged at 400 × g for 20 min. After
centrifugation, mononuclear cells were found at the plasma-61.5% Percoll layer
interface, and neutrophils were found at the 61.5-76.0% Percoll layer
interface. Neutrophils were removed and washed twice in RPMI-1640. Cells were
counted with a hemocytometer and resuspended at 106 cells/ml. Cells were plated
with the compounds investigated in the study in RPMI-1640 containing 10%
autologous-plasma-derived serum (PDS). PDS was obtained from nonanticoagulated
cell-free plasma. Cell-free plasma was obtained by centrifugation of whole
blood immediately after venipuncture. The cell-free plasma was incubated at 37°
C and PDS was derived by removal of the protein clot. The purity of neutrophils
was consistently greater than 99%.

Good luck.


Thao Nguyen wrote:

> Hi,
> I found some relevant information from the internet (bionet.immunology) on
> this subject that I am  working on yet inexperienced.  I appreciate it if
> some body can provide me helpful links, references, or your own experiences
> of a detailed procedure to isolate human neutrophils.
> Thanks
> Thao Nguyen
-------------- next part --------------
A non-text attachment was scrubbed...
Name: sam.coward.vcf
Type: text/x-vcard
Size: 319 bytes
Desc: Card for Sam Coward
Url : http://iubio.bio.indiana.edu/bionet/mm/immuno/attachments/20010802/8f796f0b/sam.coward.bin


More information about the Immuno mailing list