After 8 hours post-infection, MDDCs were harvested washing in medium and application of 0.5 mM EDTA (Gibco) in 1X PBS (Cellgro). Cell harvests were spun down, washed, and pellets resuspended in RNAlater RNA Stabilization Solution (Applied Biosystems).
CD14+ monocytes of four anonymous healthy adult female human donors (Central Indiana Regional Blood Center, Indianapolis, IN) were isolated from peripheral blood mononuclear cells by positive selection using an AutoMACS separator (Miltenyi Biotec) after isolation using lymphocyte separating media (Cellgro) following manufacturer protocols. The purity of separated monocytes was determined by flow cytometry analysis on a MCL500 flow cytometer (Beckman Coulter) using fluorescent antibody staining for CD14, CD19, CD45, and CD3 (Biolegend). Enriched monocytes were differentiated into dendritic cells (MDDC) by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF; 2,000 U/mL) and interleukin-4 (IL-4; 800 U/mL) recombinant cytokines (Peprotech) on days 0, 3, and 6 after plating in six-well plates at 2.5 × 106 cells/mL in complete RPMI medium (10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine). Immature MDDC were harvested on day 7 and plated in 6-well plates at 2 × 106 cells in 2 mL complete RPMI medium per well in the absence of exogenous cytokine. The phenotype puritiess of DCs was determined by flow cytometry analysis of CD1a, CD14, HLA-DR, CD40, CD80, and CD86 expression. All infections were performed with Leishmania major Friedlin V1 strain (NIH) and gene knockout and add-back mutants for LPG1 [encoding a galactofuranosyl transferase] or LPG2 [encoding a golgi GDP-Man transporter] genes. All parasites were cultured at 26°C without CO2 in complete medium 199 (20% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine in 50 mM HEPES, 5 mg/mL hemin in 50% triethanolamine, and 1 mg/mL biotin). Parasites tested negative for mycoplasma using a Mycoplasma PCR detection set (Takara) and tested below the detection limits for endotoxin using a Limulus Amoebocyte Lysate kit (Charles River Laboratories). Infective-stage metacyclic promastigotes were isolated by use of a Ficoll gradient as previously described (PMID: 11748963) and opsonized with 5% normal human serum prior to infection at a concentration of ten parasites to one host cell. Infection rates were determined at the end of each experiment by DiffQuick staining of cytospin whole cell preparations and visualization by light microscopy.
Total RNA from uninfected or Leishmania-infected human MDDCs was isolated using an RNEasy kit (Qiagen). Bioanalyzer 2100 (Agilent Technologies) and RNA 6000 Nano kits (Agilent Technologies) were used to determine total RNA integrity.
25ng of high quality RNA was converted to double stranded cDNA using Sigma TransPlex Complete Whole Transcriptome Amplification Kit (Sigma). RNA degradation, double stranded cDNA purification, and cDNA precipitation was conducted following NimbleGen Gene Expression Array user’s guide protocols (Roche NimbleGen). A Nanodrop ND-2000 (ThermoScientific) was used to determine total RNA and double stranded cDNA concentrations. Sample cDNAs were Cy3-labeled using NimbleGen Single Color Labeling Kit (Roche NimbleGen) per manufacturer's recommendations.
Labeled cDNAs were hybridized to 12-plex NimbleGen Homo sapiens Expression Arrays (platform accession number GPL16025), featuring 45,033 probes representing 23,752 gene transcripts, using Hybridization Kit, LS (Roche NimbleGen) and Wash Buffer Kit (Roche NimbleGen) per manufacturer's recommendations.
Image acquisition of arrays was performed using a NimbleGen MS 200 Microarray Scanner, at a 2 micron resolution.
This sample is expression values of total mRNA taken from cytokine stimulated human monocyte derived dendritic cells infected with lpg1 gene knockout add-backs Leishmania major (FV1 strain) parasites and is the fourth of four biological replicates 686_LPG1_AB
NimbleGen array image data were processed using NimbleScan (version 2.5) to extract intensity values for each gene. NimbleScan software automates the pre-processing of NimbleGen microarray image data, including identifying the location of each probe, extraction of intensity data from the image, background correction, and obtaining expression summary values for each gene using a probe-level summarization method RMA (Robust Multi-Array Average). Values are hybridization fluorescence intensity averages of RMA normalized gene transcript probesets (consisting of three probes per gene transcript). All sample values are reported for any probeset with an intensity value in at least one of the 24 samples which was two times greater than that of the corresponding sample array background. Such values were subsequently used in downstream analysis. All values for any probeset that did not pass this filter are listed as "NULL".