Affymetrix GeneChip Mouse Genome 430A 2.0 [Mouse430A_2]

Mycobacterial trehalose dimycolate reprograms macrophage global gene expression and activates matrix metalloproteinases.

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Effect of mycobacterial cell wall component TDM (trehalose dimycolate) of murine macrophage gene expression.

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Transcription profiling by array of mouse bone marrow-derived macrophages stimulated by trehalose dimycolated (TDM) or phosphatidylglycerol (PG) control to study how mycobacterial TDM reprograms macrophage global gene expression

All controls and treatments were repeated three times with RNA isolated from independent batches of primary macrophages treated with phosphatidylglycerol (PG)-coated control beads or trehalose dimycolated (TDM)-coated beads.

Mycobacterial TDM Reprograms Macrophage Global Gene Expression

Effect of mycobacterial cell wall component TDM (trehalose dimycolate) of murine macrophage gene expression.


Affymetrix:Protocol:Hybridization-Unknown

Affymetrix:Protocol:MAS4:ExpressionCall

Title: Affymetrix CHP Analysis (ExpressionCall). Description:

P-AFFY-2

Labeling of samples was carried out according to the manufacturer’s standard protocol

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

Lipid-coated microspheres were added to confluent 60 mm diameter dishes (non-tissue culture treated; Kord-Valmark) of bone marrow-derived macrophages (BMM) that had been acclimated for at least 1 h to 4 ml of fresh warmed media.  For 2 h experiments, the media was discarded and immediately replaced with 4 ml of TRIzol (Invitrogen), pipetting and rinsing to lyse the cells completely.  Lysates were immediately transferred to solvent-resistant tubes and frozen at -80 degrees C until RNA extraction.  For 24 h experiments, medium was brought up to 10 ml, 2 h after adding the microspheres, and then the cells were lysed after 24 h as described above.  RNA was extracted using the RNeasy Mini-kit (Qiagen), and then was DNase-treated using Turbo DNA-free (Ambion), following manufacturer instructions. (Parameters: Extracted product = total_RNA, Amplification = RNA polymerases)

Murine bone-marrow derived macrophages were isolated from the femurs of 5-8 week old mice (C57BL/6, 129F2, and MyD88-deficient mice on a C57BL/6/129F2 background).  Cells were differentiated in degrees Dulbecco's Modified Eagle's Medium supplemented with L-929 cell-conditioned medium (20%), heat-inactivated FCS (10%; Hyclone), L-glutamine (2 mM; Gibco), sodium pyruvate (1mM; Gibco), 100 U/ml penicillin (Gibco), and 100 ug/ml streptomycin (Gibco) for 6-7 days.  These PO cells were passaged by diluting ~1:4 and culturing for an additional 4-5 days until confluent. (Parameters: start time = 7, time unit = days, min temperature = 37, temperature unit = C, media = Dulbecco's Modified Eagle's Medium supplemented with L-929 cell-conditioned medium (20%), heat-inactivated FCS (10%; Hyclone), L-glutamine (2 mM; Gibco), sodium pyruvate (1mM; Gibco), 100 U/ml penicil)

125 ug of either M. bovis BCG trehalose dimycolate (TDM), M. tuberculosis H37Rv TDM, or PG solubilized in chloroform:methanol 2:1(v:v) were coated onto the surface of solvent-resistant tubes. 2.5 ml of a 2.5% solids solution of 90 ?m diameter polystyrene microspheres (Polysciences) for each sample were washed twice with PBS, and then coated with the lipids using alternate cycles of vortexing and sonication at 55ﾰC. Coated microspheres were washed and resuspended in endotoxin-free PBS at 2% solids. Lipid-coated microspheres were resuspended in 1 ml of PBS.  Lipid-coated microspheres were added to confluent, 60 mm diameter dishes(non-tissue culture treated; Kord-Valmark) of bone-marrow derived macrophages that had been acclimated for at least 1 hour to 4 ml of fresh warmed media. For 2 hour experiments, the medium was discarded and immediately replaced with 4 ml of TRIzol (Invitrogen), pipetting and rinsing to lyse the cells completely. Lysates were immediately transferred to solvent-resistant tubes and frozen at ?80oC until RNA extraction. For 24 h experiments, medium was brought up to 10 ml,2 hours after adding the microspheres, and then the cells were lysed after 24 hours as described above. RNA was extracted using the RNeasy Mini-kit (Qiagen), and then was DNase-treated using Turbo DNA-free (Ambion), following manufacturer instructions.

Transcription profiling by array of mouse bone marrow-derived macrophages stimulated by trehalose dimycolated (TDM) or phosphatidylglycerol (PG) control to study how mycobacterial TDM reprograms macrophage global gene expression

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