Biology Reference
In-Depth Information
2.2. Isolation of Human PMNs and Serum, and Phagocytosis Assays
1. Dextran 500 (GE Healthcare Biosciences, Piscataway, NJ).
2. Ficoll-Paque PLUS (GE Healthcare Bioscience).
3. Injection- or irrigation-grade 0.9% NaCl solution (w/v) (Hospira, Inc., Lake
Forest, IL).
4. 3% Dextran-0.9% NaCl solution (w/v). To make this solution, dissolve 30 g of
Dextran 500 into 1 L injection- or irrigation-grade 0.9% NaCl (final volume).
5. Injection- or irrigation-grade water (Abbot Laboratories, Inc., North
Chicago, IL).
6. DPBS.
7. 1.7% NaCl solution (w/v): Dissolve 17 g of RNase-free NaCl into 1 L (final
volume) of injection- or irrigation-grade water. Filter-sterilize solution using a
0.2-μm bottle top filter.
8. 50- and 250-mL conical polypropylene tubes (Corning Costar, Acton, MA).
9. 12-well flat-bottom non-pyrogenic polystyrene cell culture plates (Corning
Costar).
10. RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, CA): Prepare as
described under Subheading 2.1 .
11. 15-mL glass tube (Corning Costar) for isolating serum.
12. Limulus Amebocyte Lysate assay (Thermo Fisher, Waltham, MA). This assay
is used to periodically check reagents for the presence of endotoxin.
2.3. Purification of RNA from S. aureus
1. Lysing matrix B (0.1-mm silica spheres) (MP Biomedicals, Irvine, CA).
2. FastPrep FP120 A Instrument (MP Biomedicals).
3. RNeasy Mini Kit (Qiagen, Valencia, CA).
4. -Mercaptoethanol (-ME) (Sigma-Aldrich, St. Louis, MO).
5. Ethanol, 200-proof (Sigma-Aldrich, St. Louis, MO).
6. 1.7-mL microcentrifuge tubes, RNase-free and non-pyrogenic (Molecular
BioProducts, San Diego, CA).
7. Extra collection tubes (Qiagen).
8. Aerosol-resistant micropipette tips, RNase-free, non-stick (Ambion, Austin, TX).
2.4. Removal of Contaminating DNA from S. aureus
RNA Preparations
1. RNase-Free DNase Set, on-column digestion of DNA to be used during RNA
purification (Qiagen). Set includes DNase I (1500 Kunitz Units), DNase buffer
RDD, and nuclease-free water.
2.5. cDNA Synthesis of S. aureus RNA
1. dNTP mix: Combine 100 m M each of dATP, dCTP, dGTP, dTTP (GE Healthcare
Biosciences). For a working 10 m M dNTP mix, add 100 μL each of 100 m M
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