0 (PBS, Mediatech Inc #46-013-CM), and dispersed in cell culture complete medium for 15 minutes. ALK inhibitor Multiplicity of infection was adjusted to 10 using a standardized calibration curve of OD600/colony-forming units (cfu). Bacteria were added to host cells at 60-80% confluency in 12-well dishes. At a given timepoint after the infection, host cells were washed repeatedly with warm PBS. If indicated, remaining extra-cellular bacteria were killed by the addition of 10 μg/ml of gentamicin
to DMEM (37°C, 5% CO2) for 60 minutes. Time points given in the text for infection include this 60 minute time period of culture in the presence of gentamicin, except when infected cells were processed for immunostaining. Gentamicin was removed by washing in DMEM. Infected cells were resuspended in complete tissue culture medium without addition of antibiotics. After a given time of infection, cells were lyzed in 0.5% N-octyl LY294002 cost β-glucopyranoside (Bioscience). Serial dilutions of cell lysates were plated on Chocolate II agar and incubated
at 37°C for at two days. Infection with Salmonella was performed as described . Comparison of infection results were analyzed by the Student’s t-test, p < 0.05 was considered significant. Immunostaining Macrophage cell lines were grown on sterile coverslips in Petri dishes (6- or 12-well plates). Cells were infected with Francisella as described above, except that the step of killing extracellular bacteria with gentamicin was substituted by washing of adherent cells with DMEM three times. At indicated time points, cells on coverslips were fixed in 4% paraformaldehyde solution (Polysciences, #18814) for 10 minutes, washed with PBS and permeabilized in 0.1% Triton × 100 (Shelton Scientific IB07100) in PBS for 15 minutes. Clomifene Reaction with antisera was performed in 0.05% TWEEN20/PBS for one hour at room temperature. Stained and dried coverslips were mounted on glass slide using Gold antifade medium (Invitrogen, #P36930)
and sealed with nail polish Antiserum to TfR1 was goat polyclonal IgG (SantaCruz sc 7087), to Rab5, rabbit polyclonal IgG (Santa Cruz SC-309) and to Rab7, goat polyclonal IgG (SC11303). Antibodies were used at a dilution of 1:500. Visualization was with staining with a goat-anti-rabbit or rabbit-anti-goat IgG conjugated to Alexa 594 (Invitrogen). Microscopy A Leica AOBS laser scanning microscope was used for all fluorescence microscopy. Images were acquired using Leica software. Analyses of images was with Volocity software (Volocity 4.1 Imporvision Inc., Lexington, MA). Overlap of individual fluorescence pixels from separate channels for each optical plane was determined with the Volocity 4.1 colocalization module. When results were quantified, 100 cells from randomly selected fields were evaluated.