FITC solution was prepared 20 mg/ml in DMSO). Briefly, 1 × 109 bacteria were washed twice with 0.1 M buffer Na2CO3/NaHCO3 (pH 9) and suspended in 1 ml of the same solution. FITC was added to a final concentration of 1 mg/ml and incubated in the dark for 2 h at 37°C. Bacteria were washed gently with PBS until unbound colorant was eliminated, and used to infect J774
macrophages as was described above. Infected cells were fixed with 3% paraformaldehyde solution in PBS for 20 min and quenched by incubating with 50 mM glycine solution for 10 min. Then, cells were permeabilized with 0.05% saponin in PBS containing 0.2% BSA for 15 min, and incubated with the primary anti-LAMP-2 (ABL-93, DSHB) antibodies find more diluted 1:50 in PBS. anti-LAMP-2 antibodies Doramapimod nmr were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. Secondary antibodies anti-Rat Cy5-conjugated (Jackson Immuno Research Labs Inc.) was used diluted 1:600 in PBS. Each step with antibodies was incubated for 1 hour. Cells were mounted with Dako mounting media (Dako, Denmark)
and analysed by confocal microscopy using a Leica SP5 AOBS confocal microscope (Leica Microsystems, Germany). Internalization of the mycobacteria was followed through the fluorescence of green FITC and the LAMP-2 association to mycobacterial phagosomes was counted in at least 50 cells using Fiji/ImageJ program (U.S. National Institute of
Health, Bethesda, Maryland, USA). The analysis was performed for duplicates in three-four independent TPX-0005 experiments. Statistical determinations were made using t test. RNA preparation DNA-free RNA was extracted from 50 ml mid-exponential-phase cultures of M. tuberculosis as described by Santangelo et al. (2002) [12]. Prehybridisation, hybridisation, and washing steps were performed as described previously [13, 19]. Microarrays were hybridised with a combination of Cy3-cDNA Lumacaftor generated from genomic DNA of M. tuberculosis H37Rv and Cy5-cDNA obtained from total RNA of either M. tuberculosis H37Rv or MtΔmce2R. Eight sets of microarray data, consisting of eight biological replicates (cells from independent cultures), were produced for each M. tuberculosis strain. The microarrays were scanned using an Affymetrix 428 scanner and fluorescent spot intensities were quantified using BlueFuse for Microarrays v3.2 (BlueGnome Limited, http://www.cambridgebluegnome.com). For each spot, background fluorescence was subtracted from the average spot fluorescence to produce a channel specific ratio. Data processing and statistical analysis Log2 Cy5:Cy3 (test:control) ratios were used for subsequent calculations. Within each microarray, block median normalisation, excluding control and empty spots, was carried out using the BlueFuse software. Median absolute deviation using Mathematica 5.