In the present study, Sn expressed on primary pig macrophages was crosslinked with the anti-Sn monoclonal antibody 41D3 and the mRNA expression profiles were compared, using the Affymetrix microarray technology, to macrophages incubated with the irrelevant, isotype matched, monoclonal antibody 13D12

Transcription profiling of porcine alveolar macrophages (PAM) after antibody-mediated crosslinking of sialoadhesin (Sn, Siglec-1)

The transcriptome of porcine alveolar macrophages (PAM) after antibody-mediated crosslinking of sialoadhesin (Sn, Siglec-1)

Affymetrix GeneChip Porcine Genome Array [Porcine]

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processedDataMatrix

compound - crosslinking treatments with monoclonal antibodies

In the present study, Sn expressed on primary pig macrophages was crosslinked with the anti-Sn monoclonal antibody 41D3 and the mRNA expression profiles were compared, using the Affymetrix microarray technology, to macrophages incubated with the irrelevant, isotype matched, monoclonal antibody 13D12


P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-44455

RNA extraction and quality control RNA isolations were done using Trizol® following the manufacturers (Invitrogen Life technologies, Inc., Paisley, UK) instructions. Briefly, medium was aspirated from the cells and Trizol® (1 ml per 107 cells) was added. Cells were resuspended, transferred to an eppendorf tube and incubated for 5 min at room temperature. After that, 0.2 ml chloroform was added and the eppendorfs vigorously shaken for 15 sec, then incubated for 2-3 min at RT again. After centrifugation for 15 min at 12000 x g and at 4°C, the upper aqueous phase was transferred to a new eppendorf. RNA was precipitate following standard isopropanol-ethanol protocols. A clean-up step using QIAGEN RNeasy columns was done before proceeding to sample quantification and quality control with an Agilent 2001 Bioanalyzer (Agilent Technologies GmbH, Waldbronn, D) using RNA 6000 Nanochips. 

P-MEXP-44456

Reverse transcription (RT) of the total RNA and synthesis of biotin-labeled cRNA with one round of amplification was carried out following the standard Affymetrix one-cycle protocol according to the manufacturer's instructions. In brief, in the first strand synthesis the total RNA was reverse-transcribed with the SuperScript II reverse transcriptase (Invitrogen Life technologies) in the presence of an oligo-dT primer coupled with a primer sequence for bacterial T7-polymerase. As next, the second strand synthesis was carried out using E.coli DNA ligase, E.coli DNA Polymerase I and T4 DNA polymerase. After the synthesis of the second cDNA strand and a clean up step with phenol:chloroform:isoamyl alcohol (48:1:1), in vitro transcription (IVT) was performed and the cDNA template was transcribed with T7-polymerase in the presence of biotin-labeled ribonucleotides to yield biotin-labeled antisense cRNA. The cRNA target was fragmented and both the concentrations and the RNA quality of unfragmented and fragmented cRNA targets were determined based on OD 260/280 measurement with an Agilent 2100 Bioanalyzer.

P-MEXP-44457

P-MEXP-44458

P-MEXP-45485

The three week old piglets were injected daily with 1 ml Baytril (5% solution) and 1 ml Lincospectine/spectinomycine (5%/ 10% solution) for three days. At the age of 5 weeks, the piglets were euthanized, the PAM were collected by bronchoalveolar lavage and frozen in batches in liquid nitrogen. The Porcine alveolar macrophage (PAM) cells were grown for 24 h before treatment in Earle’s minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine (BDH Chemicals Ltd., Poole, England), 1% nonessential amino acids (Gibco-BRL, Gaithersburg, MD, USA), 1 mM sodium pyruvate and a mixture of antibiotics in a humidified 5% CO2 atmosphere at 37°C . 

P-MEXP-45486

To crosslink Sn, PAM were incubated at 37°C with either 50 µg/ml purified anti-Sn monoclonal antibody 41D3 (group A), or 50 µg/ml of the isotype-matched (immunoglobulin G1) control/mock monoclonal antibody 13D12, directed against pseudorabies virus glycoprotein gD (group B). Two hours after treatment the cells were collected in TriZol® and the RNA was extracted 

P-MEXP-45680

As first, this tool, which fits a robust linear model at the probe level and uses the RMA (Robust Multiarray Averaging) normalization method, consisting of three steps; a background adjustment, quantile normalization and final summarization [Bolstad et al. 2003; Irizarry et al. 2003] was applied to control the quality of the raw data (CEL files).For nomalization we used a AffylmGUI package (bioconductor project)[Smyth et al 2005].

Transcription profiling of porcine alveolar macrophages (PAM) after antibody-mediated crosslinking of sialoadhesin (Sn, Siglec-1)

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