Affymetrix GeneChip Bovine Genome Array Bovine

Live and inactivated Salmonella enterica serovar Typhimurium stimulate similar but distinct transcriptome profiles in bovine macrophages and dendritic cells.

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The Affymetrix bovine global microarray was used to examine the transcriptional response of Holstein-derived macrophages and dendritic cells infected with heat-inactivated Salmonella (n=6), live Salmonella (n=6) and uninfected controls (n=6) during early infection (2 hours)

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Transcription profiling of bovine macrophages and dendritic cells infected with live and heat-inactivated Salmonella

The Affymetrix bovine global microarray was used to examine the transcriptional response of Holstein-derived macrophages and dendritic cells infected with heat-inactivated Salmonella (n=6), live Salmonella (n=6) and uninfected controls (n=6) during early infection (2 hours)


Affymetrix:Protocol:Hybridization-Unknown

Affymetrix:Protocol:plier-mm

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-TABM-4557

RNA was extracted from macrophages and dendritic cells using RNasy MiniKit (Qiagen) according to manufacturer's instructions. RNA was dissolved in RNase-free water and stored in -70°C. Samples were concentrated using the Linear Acrylamide precipitation system (Ambion). The RNA concentration was measured using a Nanodrop ND100 Spectrophotometer and RNA quality was checked using Agilent 2100 bioanalyzer

P-TABM-4558

Cells were isolated from 6 female Holstein–Friesian cattle (Bos taurus) maintained at The Roslin Institute, University of Edinburgh, UK. These Holstein–Friesian cattle were between 3 and 5 years of age and kept on pasture. Dendritic cells were generated from the whole blood. The cells were separated on the MACS column with CD14+ antibody. The purity was >90% pure, as assessed by flow cytrometry with CD172alpha and CD14 staining. 1x106 cells/ml were resuspended in 3ml of DC-TCM medium (RPMI 1640 (GIBCO, USA), 10% FCS and recombinant bovine cytokines IL-4 and GM-CSF (provided by Dr Gary Entrician (Moredun Research Institute)

P-TABM-4559

Cells were isolated from 6 female Holstein–Friesian cattle (Bos taurus) maintained at The Roslin Institute, University of Edinburgh, UK. These Holstein–Friesian cattle were between 3 and 5 years of age and kept on pasture.Macrophages were isolated from whole blood.The purified PBMC were cultured in non-adherent Teflon bags (kindly provided by Prof Dirk Werling, RVC, University of London) for 7 days at 37 °C in a 5% CO2 incubator, during which time the monocytes differentiated into macrophages. Cells were resuspended in fresh medium supplemented as above, except that the FBS concentration was reduced to 2%. macrophages were purified by selective adherence.

P-TABM-4560

Initially images were quantified with the command console software followed by normalization of the data using the Plier algorithm, via Affymetrix Expression Console (version 1.0) package.Normalized log-intensities for each gene were analysed by ANOVA to generate lists of differentially expressed genes between different treatment groups. Treatment group comparisons were for differences between uninfected and infected, with dead (DI) or live bacteria (LI) for both macrophages (mF) and dendritic cells (DC). These differences were examined by modified t-test.

P-TABM-4561

Heat-inactivation of bacteria was performed by heating tubes with bacteria at 80°C for 40 minutes.  100μl of bacteria were then spread onto a LB-agar plate and left overnight at 37°C to grow.  The absence of colonies on the plate indicated successful heat-inactivation. Bacteria were diluted with 500μl serum free RPMI 1640 (GIBCO) or IMDM (GIBCO) medium and then added to the cell cultures (Mf or DC respectively).  The ratio of bacteria to APC was 50 bacteria to 1 cell.

P-TABM-4562

Salmonella enterica serovar Typhimurium wt SL1344 strain was used in this experiment. Bacteria were diluted with 500μl serum free RPMI 1640 (GIBCO) or IMDM (GIBCO) medium and then added to the cell cultures (Macrophages or DC respectively).  The ratio of bacteria to APC was 50 bacteria to 1 cell.

Transcription profiling of bovine macrophages and dendritic cells infected with live and heat-inactivated Salmonella

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