At 1 hour post infection, kanamycin (250 μg/ml) was added to kill extracellular bacteria. Cytotoxicity was measured

at 6 hr. find more post GSK2126458 molecular weight infection by assaying for lactate dehydrogenase (LDH) release in the cell supernatants using a LDH Cytotoxity Detection Kit (Clontech). Multi-nucleated giant cell assay HEK293T cells were seeded at a density of 2.5 x 104 cells/well in a 24-well tissue culture plate and infected with log-phase bacteria at MOI 10:1. Two hr. post infection, kanamycin was added to kill off extracellular bacteria and at respective time points, cells were washed with 1xPBS and fixed with 100 % methanol (Sigma-Aldrich) for 1 min. Cells were then rinsed with water and air dried before the addition of 20x diluted Giemsa stain (Sigma-Aldrich) for 20 min. After staining, cells were washed with water two times before they were air dried and examined under light microscope for MNGC formation. Cloning of full-length bopA, and bopC gene into mammalian expression vector The pcDNA3.1/V5-His TOPO (pcDNA3.1) TA Expression kit (Life Technologies) was used for cloning of full-length bopA for over-expression in mammalian systems. The bopA coding sequence including stop codon was included in the primer so that the products were not tagged. Amplified product was see more cloned into the linearized pcDNA3.1 vector according to manufacturer’s protocol. The bopC was cloned into pCMV-FLAG-MAT-Tag-1 Expression Vector (Sigma) according

to manufacturer’s instruction.

The primers for amplification of bopA and bopC are listed in Table 3. Measurement of B. pseudomallei effector gene expression by real-time PCR Total RNA was isolated from transfected HEK293T cells 24 hours post transfection using illustra RNAspin Mini Kit (GE Healthcare). cDNA was synthesized using 1 μg of RNA and the First Strand cDNA Synthesis Kit (Thermo Scientific). Transcripts were quantified using iQ Cybr Green Supermix (Bio-Rad) in a Bio-Rad iQ5 machine. The expression of effector gene was normalized to housekeeping control gene gapdh. Real-time PCR primers are listed in Table 3. Photothermal nanoblade delivery of bacteria Bacteria for photothermal nanoblade injection Ponatinib were prepared by culturing in low-salt L- broth at pH 5.8 until log-phase and then washed 3X and resuspended in Hanks balanced salt solution (HBSS) at 108–109 cfu/mL. 1–2 μl of the bacterial suspension was loaded into titanium-coated pulled-glass microcapillary pipettes. Photothermal nanoblade delivery was performed essentially as described [24, 26]. Briefly, the pulsed laser system used was a Q-switched, frequency-doubled Nd:YAG laser (Minilite I, Continuum) operated at 532 nm wavelength and 6 ns pulsewidth. The laser beam was sent into the fluorescence port of an inverted microscope (AxioObserver, Zeiss) and then through the objective lens (40X, 0.6 NA), to generate a 260 μm-wide laser spot on the sample plane. The optimized laser intensity used for bacterial delivery was 180 mJ/cm2.