Analysis of Staphylococcus aureus Gene Expression During PMN Phagocytosis - Bacterial Pathogenesis: Methods and Protocols - page 108

3. Methods
3.1. Isolation of Human Neutrophils and Serum
It is best to make certain all reagents are free of endotoxin contamination
before attempting to isolate neutrophils. This can be done using the Limulus
Amebocyte Lysate assay (as described by the manufacturer).
1. Add whole heparinized blood at a 1:1 ratio to a sterile solution of 3% dextran-
0.9% NaCl (use 50- or 250-mL tubes depending on the blood volume). Mix
gently and let stand at room temperature for 20-25 min.
2. During the dextran sedimentation, prepare the following in 50-mL conical tubes:
(1) 35 mL of 0.09% NaCl, (2) 20 mL of 1.7% NaCl, (3) 12 mL of Ficoll-
Paque PLUS , and (4) 20 mL of injection- or irrigation-grade water. These solutions
are normally stored at 4 °C, but to prevent priming and aggregation of PMNs, it
is optimal to warm the solutions to room temperature.
3. After the 20-25 min incubation, transfer the top layer from the dextran-blood
mixture to fresh tube(s) and centrifuge at 500-700 × g (with low or no break)
for 10 min at room temperature. Aspirate supernatant and discard.
4. Resuspend pellet in 10 mL of NaCl from the 35 mL prepared in step 2 . Add
remaining NaCl to 35-mL total volume.
5. Underlay 10 mL of Ficoll-Paque PLUS beneath the NaCl cell suspension. Spin at
500-700 × g (with low or no break) for 25 min at room temperature.
6. Carefully aspirate the supernatant. Using a sterile swab, wipe the inside of
the tube to remove any residual peripheral blood mononuclear cells. This step
greatly increases the purity of the PMN prep.
7. Lyse the red blood cells by resuspending the pellet in 20 mL of water. Mix
gently by pipetting for 20-30 s.
8. Immediately add 20 mL of 1.7% NaCl to prevent lysis of the PMNs and
centrifuge sample at 380 × g for 10 min at room temperature.
9. Aspirate supernatant and reususpend pellet in RPMI/H.
10. Count cells using a hemacytometer.
11. To isolate human serum, collect blood without coagulant and incubate at 37 °C
in a glass tube for 30 min. This incubation causes the whole blood to clot. After
incubation, centrifuge sample at 2000-3000 × g for 10 min. Serum can be used
immediately or stored at -20 °C for later use.
3.2. Phagocytosis of S. aureus by Human PMNs
1. Inoculate flasks of fresh tryptic soy broth (TSB) containing 5% glucose with a
1:200 dilution of an overnight culture of S. aureus . Incubate cultures at 37 °C
with shaking (250 rpm) until the optical density at 600 nm (OD 600 ) reaches 0.75.
Typically, this is mid-exponential growth phase for S. aureus . For example, an
OD 600 of 0.75 corresponds to 2.5 × 10 8 colony-forming units (CFUs)/mL for S.
aureus strain MW2 (USA400) (6) . However, the growth kinetics of S. aureus
varies between strains and depends on the growth medium used. Therefore, it