Quantifying Apoptosis and Cell Proliferation

For annexin-V and Phospho-Histone H3 (pSer^{12 (link)}) detection, the forelimb bud of embryos at time 0 or cultured for 1, 3 and 6 hours were harvested and incubated for 15 minutes in trypsin, 5% EDTA (Invitrogen). Then, tissues were disrupted mechanically and the released cells washed in complete media. For detection of annexin-V detection, cells were washed with binding buffer and incubated with annexin-V FITC (BD Pharmingen, Le Pont-de-Claix, France) during 15 minutes at room temperature. For Phospho-Histone H3 detection, cells were permeabilized with the intranuclear detection Perm/Fix solution (eBioscience) and stained 1 hour with the Phospho-Histone H3 (Ser10) antibody (Cell Signaling Technology). Then, cells were washed and stained 30 minutes with anti-rabbit Alexa 647 antibody (Cell Signaling Technology). Finally, samples were acquired on the FACS Canto II and analyzed using the FlowJo software (Ashland, OR, USA).