Infection of Human Monocyte-Derived Macrophages With Coxiella burnetii - Bacterial Pathogenesis: Methods and Protocols - page 186

Mix/DNA sample to the reaction plates. Perform real-time PCR detection of dotA
using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) or
comparable instrument. Extrapolate the number of C. burnetii genomes present
in the DNA sample from the standard curve.
4. Notes
1. Assuming a stock of approximately 3 × 10 9 C. burnetii genome equivalents per
milliliter ( see Subheading 3.5. ), this will result in a multiplicity of infection of
approximately 10 if using phase II organisms. [Phase I organisms are roughly 10-
fold less infectious for cultured cells than phase II organisms (7) .] The low ino-
culumvolume with rocking facilitates adherence and internalization of C. burnetii .
2. The Nine Mile, phase II, clone 4 isolate (RSA439) can be worked with under
biosafety level 2 laboratory conditions (27) . All other C. burnetii strains or
isolates are considered biosafety level 3 organisms. ( See CDC/NIH Biosafety in
Microbiological and Biomedical Laboratories, 4th edition.)
3. Supplementation of culture medium with 2% FBS rather than 10% retards Vero
cell growth without significantly affecting yields of C. burnetii .
4. A centrifugal force of 21,000 × g is attained using a Beckman JA14 or JLA16.250
rotor at 12,000 rpm.
5. Sonication is conducted at approximately 45 wwith a 0.5-inch horn. The sonicator
horn can be sterilized by spraying with 70% ethanol. Let air dry before use.
6. A centrifugal force of 31,000×gisattained using a Beckman JA20 or JA25.15
rotor at 16,000 rpm.
7. RenoCal-76 is chemically similar to Renografin-76 that has been historically
used to purify obligate intracellular bacteria (15) . Both contain 10% diatri-
zoate and 66% diatrizoate meglumine. Bacteria retain significant viability when
centrifuged through this medium which has a lower osmotic pressure than some
commonly used density gradient materials used to purify C. burnetii (28) .We
have also found that the Ultra-Clear centrifuge tubes are sterile if carefully
handled when removed from the box.
8. A centrifugal force of 58,400×gisattained using a Beckman SW28 rotor at
18,000 rpm.
9. This wash step will rid organisms of residual RenoCal-76.
10. Yields of approximately 1.5 × 10 10 C. burnetii genome equivalents ( see
Subheading 3.5. ) per infected T-150 flask are common.
11. A buffy coat obtained from 450 mL of blood will typically yield between 0.7
and 1.2 × 10 8 monocytes.
12. After removing the PBMC layer to a new tube, dilute the cells at least 1:2 with
PBS containing 2% FBS before centrifugation.
13. Two milliliters of RosetteSep cocktail is typically sufficient to process PBMC
from 450 mL of blood.
14. Wait until the cells begin to take on a rounded appearance and detach before
scraping.