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Cell lines and bacterial strains

U937 cells (ATCC#: CRL-1593.2) are a human monocyte lymphoma cell line ( The ExBoyfriend slim jeans AG Adriano Goldschmied Free Shipping Wholesale Price Lowest Price Cheap Online Official With Paypal Cheap Price Top Quality Online IUld2
) that exhibit macrophage-like characteristics when stimulated with phorbol myristic acid (PMA). Two srains of , (AA100 and CR39), both of which are serogroup 1 strains, were utilized in this study.

U937 cell maintenance and differentiation

U937 cells were maintained in suspension in RPMI-1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine/calf serum (FBS) and glutamine. Cells can be differentiated by the addition of 50 ng/ml PMA (5 μl of a 1 mg/ml solution of PMA per 100 ml of cells). For the purpose of electron microscopy U937 cells were either differentiated in six-well culture plates or in 12.5 cm flasks. For six-well culture plates 1×10 cells/well were seeded and incubated at 37°C with 5% CO for 48 hours in the presence of PMA. For 12.5 cm flasks, U937 cells were treated with PMA in a 175 cm flask for two days. Adherent macrophages were then removed and replated into 12.5 cm flasks and incubated at 37°C with 5% CO for 12-16 hours. Before infection U937 cells were washed three times in warm culture media.

strains and growth conditions

strain AA100 (graciously provided by Dr Yousef Abu Kwaik at the University of Kentucky, Lexington, KY) was grown on buffered charcoal yeast extract (BCYE) agar plates for 48 hours at 37°C. has previously been shown to be most virulent following logarithmic growth ( Byrne and Swanson, 1998 ). In order to achieve this growth phase, a loop full of the plate-grown bacteria was inoculated into 5 ml of pre-warmed buffered yeast extract (BYE) media in a 50 ml conical and grown at 37°C with shaking for ∼18 hours as described previously ( Crankset Reversible Shell And Ripstop Vest Patagonia Comfortable Online Free Shipping VbGmtH8Dg
). Bacterial growth was monitored spectophotometrically and bacteria were harvested once the culture OD reached 2.0-2.2 ( Gathered blouse Balenciaga Free Shipping Low Price Fee Shipping Shopping Online Reliable Cheap Price With Credit Card For Sale D6WZ7u
). An OD of 1 is equal ∼1×10/ml.

strain CR39 and its isogenic mutants were grown for 48 hours at 37°C on BCYE agar plates, then resuspended in PBS to an OD of 10.0. Bacteria from this suspension were then diluted into 12.5 cm flasks containing U937 cells to achieve the appropriate multiplicity of infection (MOI).

Infection of U937 cells with

Bacteria were suspended to the appropriate MOI in U937 culture media, in this case MOI of 20. This culture was added to the differentiated macrophages in six-well plates and spun down at 150 for 10 minutes to synchronize the infection. Plates were transferred to 37°C in air supplemented with 5% CO for the appropriate time. Extracellular bacteria were removed by washing the macrophages three times with prewarmed culture media. For longer time points, extracellular bacteria were killed using 50 μg/ml gentamicin for one hour as described previously ( Womens Jumpsuit Triangle by sOliver Outlet Websites Discount Shop For Websites Cheap Online vr7WJH790
).

Preparation of vacuoles

4.5×10 U937 cells in 50 ml of RPMI 1640 (10% FBS) were differentiated with PMA for 48 hours. were grown to an OD of 2-2.2 (∼18 hours) in BYE media, and U937 cells were infected at an MOI of 5. Infection was allowed to proceed for two hours following which cells were removed from flasks using a cell scraper in 6 ml of RPMI. 1 ml of the resuspended cells was not homogenized. The rest of the cells were homogenized in a dounce homogenizer (in/out up to five times; after each time 1 ml of cells was removed into a microfuge tube and left on ice until homogenization was complete). Microfuge tubes were spun down at 400 for three minutes to remove large fragments and/or unbroken cells (4°C). The supernatant was removed and spun down at 2000 for one minute (4°C). The pellet was resuspended in 0.5 ml fixative and spun down at 12,000 for three minutes at 4°C. Fresh fixative was added to the pellets, which were prepared for electron microscopy.

Electron microscope techniques

U937 cells were grown to confluence on either six-well culture plates or 12.5 cm plastic tissue culture flasks. that had reached the post-exponential growth phase were suspended at the appropriate MOI and added to the culture plates for the allotted time interval. At the appropriate time, for example, after 0.5, 1 or 1.5 hours, the extracellular bacteria were washed off three times with warmed culture media and the plates incubated with warmed culture media until fixation. The media was removed and the U937 cells fixed with a freshly made solution of 1% glutaraldehyde (from an 8% stock from Electron Microscopy Sciences (EMS), Fort Washington, PA) 1% OsO in 0.05 M phosphate buffer at pH 6.2 for 45 minutes. After fixation, the cells in petri plates were rinsed three times with cold distilled water and en bloc stained with uranyl acetate overnight. The petri plates were then dehydrated in ethanol then placed into hydroxypropyl methacrylate (EMS), which does not react with the plastic in the petri dish, and embedded in L 112, an epon substitute (Ladd, Burlington, VT). Following polymerization of the epon, the block was cut out and mounted and thin sections were cut through their exposed surfaces. Thin sections were collected on naked grids stained with uranyl acetate and lead citrate and examined in a Philips 200 electron microscope.

All our electron micrographs were photographed at 40,000× and printed at a magnification of 100,000×. Individual prints were selected under an illuminated dissecting microscope at a magnification of 10×. The membrane thickness was measured by placing an ocular micrometer disc on top of the segments of membranes that we wished to measure. We selected only those portions of the phagosomal membrane or the bound ER membranes where the membrane was cut transversely. Thus, after osmication one sees at one million magnification two clearly defined dense lines separating an intermediate space. If the dense lines are not clear and their margin not sharp but fuzzy, then the section is not cut perfectly normal to the membrane. Obviously these regions were not measured. On clearly defined transverse sections, we measured the width of the membrane as defined by the outer edges of the two dense lines that in electron micrographs define what we know is a membrane bilayer. To eliminate ambiguities in measurements, only one person (L.G.T.) measured all the membrane profiles. Each measurement included in Table 2 and Table 3 is of a separate ER vesicle attached to a phagosome containing a L. pneumophila bacterium. The number of separate phagosomes measured is also documented on all the tables.