Ligations were transformed into chemically competent Escherichia coli TOP10 (Invitrogen, Carlsbad, CA) and recombinant plasmids were purified using the Wizard Plus SV miniprep kit (Promega, Madison, WI).

pMoΔbsaZ was electroporated into E. coli S17-1 and mobilized into Bp K96243 as previously described [75, 76]. pMoΔbsaZ was resolved from transconjugants by culturing the isolates in LB without NaCl containing 10% (wt/vol) sucrose for 3–4 days at 25°C. Deletion of the Bp bsaZ gene was confirmed using PCR and apparent by a reduction in the amplicon size of ~1060 bp. Tissue culture and macrophage infections The RAW264.7 cell line was maintained in DMEM (Gibco) containing 10% FBS (Hyclone, Logan, UT), 1% non-essential amino acids (Sigma, St. Louis, MO), 1% selleck compound HEPES buffer (Gibco) and 1% L-Glutamine at 37°C under an INK 128 chemical structure atmosphere of 5% CO2. For macrophage infections, BD Falcon 96-well plates (Franklin

Lakes, NJ) were seeded with ~2 × 104 cells/per well and incubated overnight as described above to obtain ~4 × 104 cells/well. Macrophages were infected with Bp at a MOI of 30 (or otherwise noted) for 2 h, monolayers washed three times with PBS to remove extracellular bacteria and either macrophages were fixed (2 h infection) or pre-warmed DMEM containing 10% fetal bovine serum and 250 μg/ml of kanamycin (Sigma) Selleck OSI 906 was added to reduce extracellular bacterial growth. Infections were continued for an additional 8 h (or otherwise noted) and monolayers were fixed for ~18-24 h with 10% formalin prior to antibody staining. Macrophage

and bacterial staining Following macrophage fixation cells were washed and subsequently permeabilized for 15 minutes at room temperature with Cellomics 1× permeabilization buffer (Halethorpe, MD), washed twice with PBS and blocked (minimum of 1 h) with Cellomics 1x blocking buffer. Following incubation, blocking Protein tyrosine phosphatase buffer was removed and replaced with 50 μL of a 1:1000 dilution of 2 mg/mL anti-Burkholderia pseudomallei monoclonal antibody (AB-BURK-P-MAB3, Critical Reagents Program, Frederick, MD) for 1 h. Unbound primary antibody was removed by two washes with PBS and a 1:500 dilution of Dylight 488 goat anti-mouse secondary antibody (Fisher Scientific, Waltham, MA) was added at room temperature for 30 min. Cells were washed two additional times with PBS and 1× CellMask DeepRed (Invitrogen) and 1:10,000 Hoechst nuclear stain (Invitrogen, Carlsbad, CA) were added. Image acquisition and analysis An Opera QEHS confocal system (PerkinElmer, Waltham, MA) was used for high-throughput image acquisition. 4 imaging fields per well were acquired with a 20X water objective in the Blue (Hoechst 33342), Green (Alexa488) and Far Red (CellMask DeepRed) channels on a single Z-plane in 2 sequential exposures.