HESCs were briefly treated with Accutase for single cell and injected (~10 15 cells for each embryo) on a Nikon microscope fitted with piezo-driven Eppendorf NK2 micromanipulator, CellTram air and CellTram Vario.
Briefly, treated cells were incubated at 37 °C for 14 days and observed regularly for colony formation and the bright field images were captured with a Nikon ECLIPSE TE 2000-S inverted microscope and the number of foci were counted in random fields.
Briefly, treated cells were collected and incubated for 20 min with 5 μM JC-1 at 37 °C, washed and resuspended in media.
Human Jurkat cells were briefly treated with AKTIV or rhodamine 123 for 15 min, subjected to flow cytometry under the same conditions, and the number of fluorophores per cell was calculated based on the brightness of Cascade Blue (Φ = 0.54, ε399 nm = 28 000 M–1 cm–1) and fluorescein (ΦpH 9 = 0.93, ε490 nm = 76 900 M–1 cm–1).
Briefly, treated cells were incubated with 2.5 µM dihydroethidium for 15 min at 37°C for superoxide anion production.
Briefly, embryos were treated for 20 minutes in 1∶1 heptane:3.7% formaldehyde, hand-devitellinized, and blocked/stained in PBS/1% goat serum/0.1%TritonX-100.
Briefly, cells were treated for 72 hours with Wnt3a and confluent monolayers were scratched using a dental device to create a cell-free area where migration could be measured.
Briefly, cells were treated for 72 hours with either 1, 3 or 10 μM of 5-Azacytidine each day.
Briefly, cells were treated for 12 h with DMSO, Nut3, or 5FU, fixed with 1.0% formaldehyde and harvested for whole-cell lysate preparation in RIPA buffer.
Briefly, MEFs were treated for 1h with 0.1mM H2O2, then allowed to recover in fresh media and harvested after 1 and 6h.
