Louis, MO, USA). All parasite cultures were washed three times in a saline solution, counted, adjusted and added to macrophage cultures at a ratio of 10:1. Macrophage cultures Inflammatory peritoneal macrophages were elicited using a 3 mL intraperitoneal injection of 3% thioglycolate solution (Sigma) in C57BL/6 or CBA mice. After 96 h, all animals were
euthanized and the elicited peritoneal macrophages were obtained as previously described [3]. The cells were suspended in complete Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) [DMEM supplemented with 10% fetal bovine serum (Gibco), 2 g/L sodium bicarbonate (Sigma), 25 mM HEPES (Sigma), 1 mM glutamine (Sigma) and 0.2% ciprofloxacin (Halexistar, Goiania, GO, BR)] and distributed in 6-well plates at a concentration of 1 × 107 macrophages per well. Cultures were subsequently incubated overnight learn more at 37°C in 5% CO2. Macrophage infection The inflammatory peritoneal macrophage cultures were infected for 12 h with L. amazonensis stationary phase promastigotes. Cell cultures were then washed twice with saline to remove non-internalized
parasites and reincubated for an additional six or 24 h before either RNA extraction or fixation with ethanol for 20 min followed by staining with hematoxylin and eosin (H&E). Each independent experiment was repeated three times for microarray analysis, and each experiment was performed at least three times in triplicate for microscopic analysis. Microarray analysis Total see more RNA from uninfected or L. amazonensis-infected macrophages was prepared using Qiagen RNeasy mini-prep columns (Qiagen, Valencia, CA, USA) in accordance with manufacture protocols. The integrity of each RNA preparation was assessed using agarose gel electrophoresis. The RNA was reverse transcribed using Superscript II (Invitrogen, Carlsbad, CA, USA) in the presence of oligo(dT) primers linked to a T7 RNA polymerase promoter sequence (Proligo, La Jolla, CA, USA) to prime cDNA
synthesis. After second-strand synthesis, biotinylated cRNA was produced by in vitro transcription using biotinylated UTP and CTP (Bioarray high-yield RNA transcript labeling kit, Enzo Diagnostics, Farmingdale, NY, USA) and purified with RNAeasy mini columns (Qiagen). The biotinylated cRNA was Interleukin-2 receptor Captisol in vivo fragmented at 94°C for 30 min. For probe array hybridization and scanning, 16 μg of fragmented labeled cRNA was hybridized to the Murine Genome U74v2 GeneChip® array (Affymetrix, Santa Clara, CA, USA), which contains nearly 400,000 probe sets covering approximately 12,000 different murine genes. Array scanning was performed using the Affymetrix® GeneChip Scanner 3000 7 G and all images were analyzed using Microarray Analysis Software (Affymetrix v5.0). Experimental data are available online at ArrayExpress (E-MEXP-3448).