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. Author manuscript; available in PMC: 2014 Feb 18.
Published in final edited form as: Nat Protoc. 2013 May 2;8(6):1028–1041. doi: 10.1038/nprot.2013.049

Figure 4.

Figure 4

Timeline for XEN cell derivation from mouse blastocysts. Protocol for the derivation of XEN cells from blastocysts. Day 0: feeder cell–coated four-well plates are prepared. Day 1: medium is replaced and a freshly flushed E3.5 blastocyst is placed in the center of the well. Day 2: the blastocyst hatches and attaches to the feeder cells. Day 3: the blastocyst has formed an outgrowth. Medium is replaced. Day 4: the prominent outgrowth is trypsinized and disaggregated with a P20 pipette. Days 5–10: the medium is replaced every other day and the plate is observed daily. Days 10–15: XEN-like cells with stellate and refractile morphology will emerge. The medium is replaced every other day and the plate is observed daily. Day 15 and onward: After 70% confluency is attained, the XEN cells are passaged onto a well in a six-well plate and subsequently into a 100-mm dish. After three more passages they can be MEF-depleted and the XEN cell line can be frozen. Scale bars, 100 µm.