HUES-2 ES cells were cultured at 37_C, 5% CO2 and atmospheric O2, and maintained on mitomycin C-inactivated mouse embryo fibroblasts (MEF) in 0.1% gelatin-coated six-well tissue culture plates containing 2 mL hES medium. hES medium consisted of knockout Dulbecco’s modified Eagle’s medium (Invitrogen GIBCO), 10% KO-Serum Replacement (Invitrogen GIBCO), 0.5% albumin (cat. no. 534021-4l; Sigma, St Louis, MO, USA), 2 mM Glutamax-I (Invitrogen GIBCO), 1% nonessential aminoacids (Invitrogen GIBCO), 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen GIBCO), 0.055 mM b-mercaptoethanol, 10 ng/mL basic fibroblast growth factor (R&D). Medium was changed daily. Differentiation of hES cells into monocytes: For embryoid body (EB) formation, hES cells were removed from feeder cells either mechanically by microdissecting hES colonies into 0.2-mm–diameter patches. Patches were transferred into six-well ultra-low adherence plates (Corning) in hES culture medium and cultured for 3 days at 37_C. For differentiation to monocytes, 50 to 100 EBs were transferred into tissue culture T25 flasks containing 10 mL culture medium supplemented with M-CSF and IL-3. Medium was replaced every 5 to 7 days. Culture medium consisted of Advanced Dulbecco’s modified Eagle’s medium (Invitrogen GIBCO) and 10% fetal calf serum (Invitrogen GIBCO or Hyclone), supplemented with 50 ng/mL M-CSF (R&D), 25 ng/mL IL-3 (R&D), 2 mM L-glutamine (Invitrogen GIBCO), 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen GIBCO), and 0.055 mM b-mercaptoethanol. HUES2 Human ES cell-derived monocytes were differentiated into macrophages at a density of 1.5 _ 105 cells/cm2. Culture medium consisted of RPMI (Invitrogen GIBCO) and 10% fetal calf serum (Hyclone), supplemented with 100 ng/mL M-CSF (R&D), 2 mM L-glutamine (Invitrogen GIBCO), 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen GIBCO). Medium was changed at day 3. Cells were harvested at day 7, by washing with PBS then lysing adherent cells in RNeasy lysis buffer (Qiagen). Adult human blood was obtained from anonymous donors through the UK National Blood Bank Service, and tested negative for HIV-1, hepatitis B/C, and syphilis. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque (Pharmacia-Amersham) density gradient centrifugation from heparinized buffy coats. Monocytes were isolated by CD14-positive selection using anti-CD14 magnetic beads (Miltenyi Biotec) by following manufacturer’s instructions.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted with the RNeasy minikit (Qiagen).
Label
biotin
Label protocol
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
Hybridization protocol
Samples were hybridized using Affymetrix hybridization kit materials • Heat cocktails at 99° for 5 minutes, then 42° for 5 minutes centrifuge at max speed for 1 minute (N.B. this deviates from Affy SOP). • Transfer 200μl of hyb solution to each array, then tape holes and parafilm. • Hybridize 16 hours at 45° at 60rpm• Fluidics washing is FS450_0001
Signal values were generated using the Robust Multichip Average method with the Affymetrix Expression Console software. Statistical analysis and hierarchical clustering were done using the TM4 software.
