Ibitreat microscopy plastic μ plates

E3.5 embryos were recovered by flushing uteri with M2 medium. Zona pellucida were removed by brief exposure to acidic Tyrode’s solution (Sigma). Zona-freed blastocysts were seeded on ibiTreat microscopy plastic μ plates (Ibidi), filled with prewarmed IVC1 medium (Advanced DMEM/F12 (GIBCO) containing 20% FCS (Biosera) and supplemented with 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), penicillin (25 units/ml)/streptomycin (25 μg/ml) (GIBCO,), 1 × ITS-X (Invitrogen), 8 nM β-estradiol (Sigma), 200 ng/ml progesterone (Sigma), and 25 μM N-acetyl-L-cysteine (Sigma). In the following 36–48 hr, embryos attached to the surface of the plate as TE differentiated into giant cells. The medium was then exchanged and emerging egg cylinders cultured in serum-free, chemically defined IVC2 medium (Advanced DMEM/F12 (GIBCO) containing 30% KSR (KnockOut Serum Replacement, GIBCO) and supplemented with 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), penicillin (25 units/ml)/streptomycin (25 μg/ml) (GIBCO), 1 × ITS-X (Invitrogen), 8 nM β-estradiol (Sigma), 200 ng/ml progesterone (Sigma), and 25 μM N-acetyl-L-cysteine (Sigma). Embryo culture was performed at 37°C in 5% CO₂. Procedures used for imaging living or fixed preparations of cultured embryos are given in the Extended Experimental Procedures.

ICMs of E3.5 embryos were isolated by immunosurgery (Solter and Knowles, 1975 (link)). Individual ICMs were placed in drops of matrigel in ibiTreat microscopy plastic μ plates (Ibidi) and incubated at 37°C for 2–3 min until the matrigel solidified. The plate was then filled with warmed IVC1 medium and ICMs cultured for 3 days at 37°C in 5% CO₂.

ES cells were washed once with PBS and were incubated for 10 min at 37°C in 0.05% trypsin-EDTA (Invitrogen). Cells were pelleted by centrifugation for 5 min/1,000 rpm, washed with PBS, and repelleted. The pellet was resuspended in matrigel (BD, 356230) by pipetting to single cells. The cell suspension was plated on ibiTreat microscopy plastic μ plates (Ibidi) and incubated for 2–3 min on 37°C until the matrigel solidified. The plate was then filled with prewarmed medium (DMEM, GIBCO) supplemented with 15% FCS (Sigma), 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), penicillin 50 units/ml/streptomycin 50 μg/ml (GIBCO), 1 × NEAA (GIBCO), and 100 mM β-mercaptoethanol. Cells were cultured at 37°C and 5% CO₂. In control experiments, ES cells were cultured in 1% low melting point agarose instead of matrigel. ES cells were isolated from E3.5 CAG-GFP embryos as previously described (Stemmler and Bedzhov, 2010 (link)). β1-integrin null and control wild-type ES cells were the kind gift of Reinhard Faessler.