At t=24h, amastigotes were NOT added to bone marrow-derived macrophages. Parasite-free macrophages were cultured at 34°C for 24h.
Growth protocol
Bone marrow cell suspensions recovered from tibias and femurs of BALB/c mice were suspended in RPMI 1640 medium (Seromed) supplemented with 10% FCS, antibiotics and 20% L-929 fibroblast-conditioned medium. Cells were distributed in bacteriologic Petri dishes (Greiner, Germany) and incubated at 37 °C in a 5% CO2 air atmosphere. Six days later, bone marrow-derived smoothly adherent macrophages (BMDMs) were washed with Dulbecco’s phosphate buffered solution (PBS) and resuspended in prewarmed 1% EDTA in Dulbecco’s PBS without Ca2+ and Mg2+ (Biochrom AG, Berlin, Germany). Recovered BMDMs were deposited in flat-bottom 24-well plates (Tanner, Switzerland) at a density of 5 * 10e-05 cells per well (reference time: t=0h).
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction directly from living Macrophages(RNeasy+ Mini-Kit, Qiagen), RNA quality control and concentration were determined using the NanoDrop ND-1000 microspectrophotometer. RNA integrity was assessed using the Agilent-2100 Bioanalyzer (RNA Integrity Numbers ≥ 9/10).
Label
biotin
Label protocol
Affymetrix standard protocol: www.affymetrix.com/support/technical/other/oncecyle_presentation.pdf
Hybridization protocol
Affymetrix EukGE-WS2v5 Hybridization
Scan protocol
Standard Affymetrix protocol
Description
None
Data processing
Data processing, background correction, normalization and signal quantification were carried out with GC-Robust Multiarray Analysis (GC-RMA) algorithm using all samples (I1, I2, UI1 and UI2). Unreliable probe-sets called “Absent” by Affymetrix GeneChip Operating Software (GCOS, www.affymetrix.com/support/technical/manual/expression_manual.affx) for at least 3 out of 4 GeneChips were discarded from subsequent analyses, as well as probe-sets called “Absent” once within samples plus once within controls (total number of probe-sets filtered out: 25,994 out of 45,101, i.e. 57.64%; see online supplementary material). Local pooled error (LPE) tests were performed to identify significant differences in gene expression between parasite-free and parasite-harboring MΦs. Benjamini-Hochberg (BH) multiple-test correction was applied to control for the number of false positives with an adjusted 5% statistical significance threshold. We used GC-RMA algorithm, LPE tests and BH multiple-test correction as implemented in Splus ArrayAnalyzer® module (www.insightful.com/products/s-plus_arrayanalyzer/). A total of 1,248 probe-sets showed significant differential expression, and were input into Ingenuity Pathway Analysis software 5.5.1 (www.ingenuity.com) to perform a biological interaction network analysis.