Excitation, long pass, and band pass wavelengths were 488 nm, 635 nm, and 695 +/- 40 nm, respectively. Upon completion of FACS, the volume of the sorted cells (about 1 ml) was immediately adjusted to 12 ml with BSK-II and incubated at 34°C. The FlowJo program suite, version 7.2.2 (Treestar), was used for data analysis. DNA sequence analysis and identity of subsurface retention signals Spirochetes were counted using a Petroff-Hauser counting chamber, adjusted to 200 cells ml-1, plated on solid BSK II media [12], and incubated at 34°C and 5% CO2. Individual colonies were picked using sterile toothpicks and cultured in 200 μl of BSK-II

complete media in a sterile 96-well tissue culture plate (Corning). The mutated ospA-mrfp1 region was amplified from 1 μl of 1:10 diluted culture in sterile water using primers Mutscreen-fwd and -rev (Figure 1 and Table 1). PCR products were purified using a PCR purification AG-881 supplier kit (Qiagen) and sequenced (AGCT Inc., Wheeling, IL) using primer Mutscreen-seq. Each sequenced mutant was cultured in liquid BSK-II culture for further analysis. Protein localization assays To assess https://www.selleckchem.com/products/epz015666.html protein surface exposure by protease accessibility intact B. burgdorferi cells were treated in situ with proteinase K as described [4, 15].

In order to determine localization of mRFP1 outer membrane vesicles were isolated and purified by treatment of B. burgdorferi cells with low pH, hypotonic citrate buffer followed by isopycnic sucrose gradient ultracentrifugation as described [4, 16]. Protein gel electrophoresis and immunoblot analysis Proteins were separated by sodium dodecyl sulfate-12.5% or -10% polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie blue staining. For immunoblots, proteins were electrophoretically transferred to a Immobilon-NC nitrocellulose membrane (Millipore) using a Transblot semi-dry transfer cell (Bio-Rad) as described. Membranes were rinsed in 20 mM Tris-500 mM NaCl, pH 7.5 (TBS). TBS with 0.05% Tween 20 (TBST) containing 5% dry milk was used for membrane blocking and subsequent

Amisulpride incubation with primary and secondary antibodies; TBST alone was used for the intervening washes. Antibodies used were anti-mRFP1 rabbit polyclonal antiserum ([17]; 1:5000 dilution, a gift from P. Viollier, Case Western Reserve University, Cleveland, OH), anti-OppAIV rabbit polyclonal antiserum ([18]; 1:100 dilution, a gift from P.A. Rosa, NIH/NIAID Rocky Mountain Laboratories, Hamilton, MT) and anti-FlaB rabbit polyclonal antiserum ([19]; 1:1000 dilution; a gift from M. HSP inhibitor Caimano, Univ. of Connecticut Health Center, Farmington, CT), or anti-OspA mouse monoclonal ([20]; H5332; 1:50 dilution). Secondary antibodies were alkaline phosphatase-conjugated goat anti-rabbit IgG (H+L) or goat anti-mouse IgG (H+L) (Sigma).