Affymetrix GeneChip Human Genome HG-U133A [HG-U133A]

Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages

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Human monocytes were purified from cytapheresis rings and differentiated into macrophages or DCs according to a previously described procedure

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Transcription profiling by array of human dendritic cells and macrophages infected with Mycobacterium tuberculosis

Transcription profiling of human DC and macrophages stimulated with Mycobacterium Tuberculosis

Human monocytes were purified from cytapheresis rings and differentiated into macrophages or DCs according to a previously described procedure


Affymetrix:Protocol:Hybridization-Unknown

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

Before infection, M. tuberculosis H37Rv was rapidly thawed and incubated overnight in 30 ml of 7H9 with glycerol and hygromycin. Bacteria were then washed three times with and resuspended in 1 ml RPMI 1640. Clumps were disassociated by 25-30 passages through a needle, followed by 5 min of sedimentation. The density of bacteria in the supernatant was checked at OD600 and correlated to the numeration of the aliquot to allow 1-to-1 bacterium-per-cell infection. This was performed in six-well plates with 2 x 106 cells in 3 ml medium containing the respective cytokines for macrophages and DCs. After 6 h of incubation at 37 C, infected cells were washed three times in RPMI 1640 to remove extracellular bacteria and were incubated in fresh medium for a further 1-2 days. In experiments on the antimycobacterial effect of GM-CSF, M-CSF was replaced by GM-CSF 18 h before infection of M?s. Unless otherwise indicated, cytokines were maintained in culture throughout the course of infection. After 2 days of infection, the percentages of dead cells (dextran blue exclusion assay) were 25-40% for DCs and 50-75% for macrophages.

Transcription profiling by array of human dendritic cells and macrophages infected with Mycobacterium tuberculosis

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