Infection of Human Monocyte-Derived Macrophages With Coxiella burnetii - Bacterial Pathogenesis: Methods and Protocols - page 182

system. Treatment of monocytes with M-CSF results in their differentiation into
cells with typical macrophage morphology that efficiently ingest and support
the growth of C. burnetii .
3.1. Infection of Vero Cells with Coxiella burnetii
1. Passage confluent Vero cells in a single T-150 flask 1:12 into 12 T-150 flasks.
This split will provide confluent monolayers in new flasks for C. burnetii infection
in 24-48 h. Grow cells in RPMI medium with 10% FBS at 37 °C in 5% CO 2 .
2. Quick thaw a C. burnetii seed stock in a 37 °C water bath, then place on ice. In
a 50-mL conical tube, make 48 mL of inoculum consisting of a 1:50 dilution of
the seed stock in RPMI with 2% FBS. Discard the tissue culture media and add 4
mL of inoculum per T-150 flask. Incubate for2hatroom temperature with slow
rocking ( see Notes 1 and 2 ).
3. Add 40 mL of RPMI with 2% FBS directly to flasks without removing inoculum
( see Note 3 ). Incubate for approximately 7-10 days at 37 °C in 5% CO 2 .At
this time point, C. burnetii will be in its stationary growth phase, and the yield
of organisms will be maximized (20) . Large, usually single replicative vacuoles
become apparent beginning at approximately 2 days post-infection ( see Fig. 1 ).
Fig. 1. Vero cells infected with the Nine Mile phase I strain (RSA493) of Coxiella
burnetii . Vero cells in a 24-well plate were infected for 5 days, then viewed by phase
contrast light microscopy using a Nikon TE-2000E inverted microscope equipped with
a CoolSNAP HQ digital camera (Roper Scientific, Tuscon, AZ). Images were acquired
using Metamorph software (Universal Imaging, Dowingtown, PA) and processed using
Adobe Photoshop (Adobe Systems, San Jose, CA). Vero cells containing large and usually
singular replicative vacuoles harboring C. burnetii are evident (arrow). Bar, 25 μm.