Infection of Human Monocyte-Derived Macrophages With Coxiella burnetii - Bacterial Pathogenesis: Methods and Protocols - page 180

2. African green monkey kidney epithelial (Vero) cells (CCL-81; American Type
Culture Collection, Manassas, VA).
3. Phosphate-buffered saline (PBS): Na 2 HPO 4 , 53.9 m M ;KH 2 PO 4 , 12.8 m M ; and
NaCl, 72.6 m M . Sterilize by autoclaving and store at room temperature.
4. PBS-sucrose (PBSS): PBS containing 0.25 M sucrose. Sterilize by passage
through a 0.22-μm Millipore filter and store at room temperature.
5. 30% (v/v) RenoCal-76 (Bracco Diagnostics Inc, Princeton, NJ) in PBSS. Store
at 4 °C.
6. 150-cm 2 cell-culture flasks (Corning, Corning, NY).
7. Cell scrapers (39-cm handle, 3-cm blade) (Corning).
8. 50-mL disposable conical centrifuge tubes (Corning).
9. Sterile 50 mL O-ringed screw cap centrifuge tubes (Nalge Nunc International,
Rochester, NY).
10. Sterile 250-mL O-ringed screw cap centrifuge bottles (Nalge Nunc International).
11. Ultra-Clear centrifuge tubes (25 mm× 89 mm) (Beckman Coulter, Fullerton, CA).
12. Ethanol (70%).
13. 10-mLLuer-lock Tip disposable syringes (BectonDickinson, Franklin Lakes, NJ).
14. 14-gauge, 4-inch metal cannulas (Popper and Sons, Inc., New Hyde Park, NY).
15. Sterile 8-ounce disposable plastic beakers (Daigger and Company, Vernon
Hills, IL).
16. Sterile 2-mL O-ring screw cap microfuge tubes (Sarstedt AG and Co.,
Numbrecht, Germany).
2.2. PBMC Isolation and MDM Culture
1. Ficoll-Paque Plus (Amersham Pharmacia, Piscataway, NJ). Store at room temper-
ature and protect from light.
2. PBS supplemented with 2% FBS.
3. RosetteSep Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver,
Canada).
4. RPMI medium with Glutamax I, supplemented with 10% FBS (Invitrogen). Store
at 4 °C.
5. Macrophage colony-stimulating factor (M-CSF) (Peprotech, Rocky Hill, NJ)
dissolved in PBS plus 2% FBS to make a 50 μg/mL stock. Stable for weeks
at -20 °C or months at -80 °C.
6. 150-cm 2 cell-culture flasks (Corning).
7. Cell scrapers (39-cm handle, 3-cm blade) (Corning).
8. 25-mL pipettes (BD Falcon, Balford, MA).
2.3. Quantitative PCR Coxiella burnetii Growth Assay
1. Ultraclean Microbial DNA isolation kit (MoBio, Carlsbad, CA).
2. TaqMan Master Mix solution (Applied Biosystems, Foster City, CA).
3. PCR primers and probe corresponding to C. burnetii dotA : dotA-F (5´-GCGCAA
TACGCTCAATCACA-3´), dotA-R (5´-CCATGGCCCCAATTCTCTT-3´), and