7 protocols
AccessionNameType
P-AFFY-6
feature_extraction
Title: Affymetrix CEL analysis. Description:
Affymetrix:Protocol:Hybridization-Unknown
hybridization
P-AFFY-2
labeling
Affymetrix:Protocol:MAS4:ExpressionCall
bioassay_data_transformation
Title: Affymetrix CHP Analysis (ExpressionCall). Description:
P-MTAB-30682
grow
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)
P-MTAB-30683
specified_biomaterial_action
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 55°C. 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.
P-MTAB-30681
nucleic_acid_extraction
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)