Infection of Human Monocyte-Derived Macrophages With Coxiella burnetii - Bacterial Pathogenesis: Methods and Protocols - page 183

3.2. Purification of Coxiella burnetii from Vero Cells
1. Detach infected monolayer into culture medium by scraping with a cell scraper
Transfer equal amounts of medium containing detached cells into two sterile
250-mL centrifuge bottles. Serially, rinse flasks with 10 mL of PBSS and add an
equal amount of rinse to each centrifuge bottle. Centrifuge for 15 min at 21,000
× g at 4 °C. This centrifugation step pellets not only infected host cells but also
C. burnetii present in the culture medium ( see Note 4) .
2. Discard supernatant. Combine pellets in approximately 30 mL of cold PBSS. Use
a 10-mL syringe and sterile cannula to resuspend and disperse the pellet. Transfer
resuspended organisms to a sterile disposable plastic beaker and place on ice.
3. Sonicate for 15 s at medium setting to lyse infected Vero cells. Cool on ice for
30 s and then sonicate for an additional 15 s ( see Note 5 ).
4. Transfer material to a 50-mL conical centrifuge tube. Centrifuge at 900 × g for 5
min at 4 °C to pellet unbroken cells and nuclei. Transfer post-nuclear supernatant
containing C. burnetii to a 50-mL O-ringed screw cap centrifuge tube.
5. Centrifuge sample for 15 min at 31,000 × g at 4 °C to pellet C. burnetii ( see Note
6 ). Discard supernatant. Resuspend pellet in 20 mL of cold PBSS using a 10-mL
syringe and cannula.
6. Gently layer 10 mL of the C. burnetii suspension equally over two 8-mL 30%-
RenoCal-76 cushions in 25 × 89 Ultra-Clear centrifuge tubes ( see Note 7 ). Fill
tubes with PBSS. Centrifuge for 30 min at 58,400 × g at 4 °C. C. burnetii will
move through the pad to form a whitish pellet while most host cell material will
remain at the RenoCal-76/PBSS interface ( see Note 8 ).
7. Gently pour off the supernatant. Resuspend each pellet with 10 mL of cold PBSS
using a 10-mL syringe and cannula. Transfer each 10-mL resuspension to a 50-mL
O-ringed screw cap centrifuge tube. Fill tubes with PBSS. Centrifuge sample for
15 min at 31,000 × g at 4 °C to pellet C. burnetii ( see Note 9 ).
8. Discard supernatant. Resuspend and combine pellets in 6 mL of PBSS using a
10-mL syringe and cannula. Aliquot purified C. burnetii in volumes of 0.1-1.0
mL in 2-mL microfuge tubes. Store at -80 °C ( see Note 10 ).

3.3. PBMC Isolation and Culture of Monocyte-Derived Macrophages
1. Prior to starting, set aside at least 3 mL of whole blood from the donor for use in
the next step. Dilute whole blood or buffy coat (highly enriched for leukocytes but
still containing a large number of erythrocytes) 1:2 with PBS containing 2% FBS
( see Note 11 ). Carefully layer 25 mL of diluted blood onto 20 mL of Ficoll-Paque
Plus in a 50-mL conical tube. Centrifuge for 30 min at 500 × g with the brake off.
After centrifugation, a layer of PBMC will be found at the Ficoll-diluted blood
interface. Carefully remove this layer with a 25-mL pipette and transfer to a clean
50-mL conical tube ( see Note 12 ). To remove platelets, wash PBMC twice with
PBS containing 2% FBS by centrifuging at 200 × g in between each wash step.
2. Resuspend PBMC pellet in PBS with 2% FBS, adjusting the cell concentration
to 2-5 ×10 7 cells/mL. For every 25 mL of PBMC solution, add 3.3 mL of