Human Primary Neutrophils. We isolated human primary neutrophils from discarded white blood
cell filters (WBF2 filter; Pall Corporation, East Hills, NY),
which were provided by the Blood Bank Lab at Children's Hospital
Boston. Neutrophils are purified by using a standard protocol.
Briefly, erythrocytes were sedimented by adding an equal volume
of dextran/saline solution (3% dextran T-500 in 0.9% NaCl) at
room temperature for 25 min. The erythrocyte-depleted supernatants
then were layered on Lymphocyte Separation Medium (1.077 g/ml
Ficoll-Hypaque solution; Voigt Global Distribution LLC, Kansas
City, MO) and centrifuged at 400
x g at room temperature for
20 min. Contaminated erythrocytes in the neutrophil pellets
were lysed after a brief (<30 seconds) treatment with 0.2%
NaCl. Neutrophils then were resuspended in RPMI medium 1640
containing 10% heat-inactivated FBS at a density of 4
x 10
6cells per ml and maintained at 37°C. The purity of neutrophils
was >97% as determined by both Wright–Giemsa staining
and FACS analysis with CD15 antibody (data not shown). We routinely
obtained
1–3
x 10
8 neutrophils from one filter (450 ml
of blood from a healthy donor). We compared the neutrophils
that we collected through filter with those obtained by vein
puncture and stored in anticoagulant testing tubs and found
that the filtration method does not impair neutrophil function
(e.g., chemotaxis and the time course of cell death). All blood
was drawn from healthy blood donors. All protocols were approved
by the Children's Hospital Institutional Review Board and were
subjected to annual review.
Mouse Neutrophils. The conditional Pten knockout mouse (PTEN loxP/loxP) and themyeloid-specific Cre mouse were purchased from The Jackson Laboratory(Bar Harbor, ME). Mouse genotyping was performed by using astandard protocol provided by The Jackson Laboratory. Mouseneutrophils were prepared from bone marrow, which was isolatedfrom the femur and the tibia of 10-week-old mice. Bone marrowneutrophils are separated by centrifugation over a three-layerPercoll gradient (78%/69%/52%). We routinely obtain 7–8million cells from one mouse, and >90% of them are morphologicallymature neutrophils (donut-shape, segmented nucleus). All animalmanipulations were conducted in accordance with the Animal WelfareGuidelines of Children's Hospital and were monitored by theChildren's Hospital Animal Care and Use Committee. To generatemature neutrophils in vitro, 4 million total bone marrow cellswere cultured in semisolid METHOCULT GF M3534 medium (StemCellTech, Vancouver, BC, Canada) in which only monocytes and granulocytecolonies can be formed. We can routinely obtain 40–60granulocyte colonies from one plate. Neutrophil death in thesecolonies was analyzed as described below.
FACS Analysis of Neutrophil Spontaneous Death. Neutrophils were cultured for an indicated time and stainedby using an Annexin V Detection Kit (Caltag Laboratories, Burlingame,CA) following a protocol provided by the manufacturer. FACSwas performed by using a FACScan flow cytometer (Becton Dickinson,San Jose, CA) equipped with a 488-nm argon laser. Ten thousandcells were collected and analyzed by using the CellQuest software(Becton Dickinson).
Cell Lines and Cell Death Assays. HeLa cells, a human cervical carcinoma-derived cell line, andHEK293 cells, a kidney tumor cell line, were maintained in DMEMwith 10% FBS, 2 mM L-glutamine, and 100 units of penicillin–streptomycinat 37°C with 5% CO2 atmosphere in a humidified incubator.Cell death was induced by the addition of 1 mM H2O2 or 1 µMstaurosporine. Toxicity was assayed 12–14 h after drugexposure by microscopic examination with computer-assisted cellcounting. For staining of dead cells by TUNEL assay, cells werefixed in 4% paraformaldehyde/PBS and then stained by using aTUNEL Assay Kit by following protocols provided by the manufacturer(Molecular Probes, Eugene, OR). The broad-spectrum caspase inhibitor,zVAD-fmk (10 µM), was added 1 h before drug treatments.Cell death was determined as the ratio of dead (TUNEL-positive)-to-totalcell number and quantified by counting 1,000 cells. Westernblot analysis was conduced as described in ref. 33.
Statistical Analysis. Values shown in each figure represent mean ± SD. Statisticalsignificances were calculated with the Student t test. Differenceswere considered significant for P values <0.005.
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