Human embryos cryopreserved at the blastocyst stage were thawed by a two-step rapid thawing protocol using Quinn's Advantage Thaw Kit (CooperSurgical, Trumbull, CT) as previously described35 ,36 . In brief, either cryostraws or vials were removed from the liquid nitrogen and exposed to air before incubating in a 37 °C water bath. Once thawed, embryos were transferred to a 0.5 mol l−1sucrose solution for 10 min followed by a 0.2 mol l−sucrose solution for an additional 10 min. The embryos were then washed in Quinn’s Advantage Medium with Hepes (CooperSurgical) plus 5% serum protein substitute (CooperSurgical) and each transferred to a 25 µl microdrop of either Quinn’s advantage cleavage medium (CooperSurgical) or Quinn’s advantage cleavage medium (CooperSurgical) supplemented with 10% serum protein substitute under mineral oil (Sigma, St Louis, MO). The embryos were cultured at 37 °C with 6% CO2, 5% O2 and 89% N2 under standard human embryo culture conditions in accordance with current clinical IVF practice. Embryos used in this study were days post fertilization (DPF) 5–6.
Serum protein substitute
Manufactured by CooperSurgical
Sourced in United States
Serum protein substitute is a laboratory product designed to replace serum proteins in various experimental and analytical procedures. It provides a consistent and standardized protein source for use in cell culture, immunoassays, and other applications where serum proteins are required but need to be controlled or reduced.
Automatically generated - may contain errors
Lab products found in correlation
Quinn s advantage cleavage medium, by CooperSurgical (4 mentions)
Quinn s advantage thaw kit, by CooperSurgical (3 mentions)
Serum protein substitute, by Merck Group (2 mentions)
Quinn s advantage medium with hepes, by CooperSurgical (2 mentions)
Mineral oil, by Merck Group (2 mentions)
Quinn s advantage serum protein substitute, by CooperSurgical (1 mentions)
Hepes, by CooperSurgical (1 mentions)
5 protocols using serum protein substitute
1
Rapid Thawing and Culture of Human Blastocysts
2
Thawing and Culturing Frozen Human Embryos
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Frozen human embryos at different stages of preimplantation development from zygotic up to blastocyst stage were thawed as described previously (Wong et al. 2010 (link)). Briefly, cryo-containers were removed from liquid nitrogen and exposed to air for 10 s before incubating in a water bath at 37 °C until thawed. Next, embryos were transferred to 0.5 M and 0.2 M sucrose solution for 10 min each and washed in diluent solution for 10 min at room temperature (RT). The embryos were then incubated in Quinn’s Advantage cleavage medium (CooperSurgical, Trumbull, CT, USA) supplemented with 10% serum protein substitute (CooperSurgical, Trumbull, CT, USA) under mineral oil (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 6% CO2, 5% O2, and 89% N2, standard human embryo culture conditions in accordance with current clinical IVF practice.
3
Single-Cell Isolation from Human Embryos
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Human embryos were cultured as described previously 30 , 31 . Thawed embryos were placed in a polystyrene dish containing 0.5 M sucrose solution for 10 minutes, then 0.2 M sucrose solution for a subsequent 10 minutes. Next, embryos were washed with Quinn's Advantage Medium with HEPES (Cooper Surgical, Trumbull, CT, http://www.coopersurgical.com ) with the addition of 5% Quinn's Advantage Serum Protein Substitute (Cooper Surgical). Embryos were cultured in either Quinn's Advantage Cleavage or Blastocyst Medium (depending on stage) plus 10% Serum Protein Substitute (Cooper Surgical) under mineral oil (Sigma, St Louis, MO, http://www.sigmaaldrich.com ) at 37°C with 6% CO2, 5% O2, and 89% N2 under standard human embryo culture conditions and in agreement with current clinical IVF practice. The zona pellucida was removed by treatment with Acidified Tyrode's Solution (Millipore, Billerica, MA, http://www.millipore.com ), and single blastomeres were collected by incubating in Quinn's Advantage Ca2+ and Mg2+‐free medium with HEPES (Cooper Surgical) for 5–20 minutes at 37°C with pipetting to break up into single cells. Blastomeres were tubed and flash frozen at −80°C until qRT‐PCR analysis.
4
Thawing and Culturing Cryopreserved Human Embryos
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Human embryos were thawed using a Quinn's Advantage Thaw Kit (CooperSurgical) as described before26 (link)27 (link). In brief, cryovials or straws were removed from the liquid nitrogen tank and exposed to air before being placed in a 37°C water bath. Once thawed, embryos were incubated in 0.5 and 0.2 M sucrose thawing medium at 37 °C for 10 min each. The embryos were then washed in thaw diluent solution at 37 °C and cultured for 3–4 h in Quinn's advantage cleavage medium (CooperSurgical) supplemented with 10% serum protein substitute (CooperSurgical). In some experiments, embryos were monitored for their development using the Eeva System (Auxogyn).
5
Rapid Thawing and Culture of Human Blastocysts
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Human embryos cryopreserved at the blastocyst stage were thawed by a two-step rapid thawing protocol using Quinn's Advantage Thaw Kit (CooperSurgical, Trumbull, CT) as previously described35 ,36 . In brief, either cryostraws or vials were removed from the liquid nitrogen and exposed to air before incubating in a 37 °C water bath. Once thawed, embryos were transferred to a 0.5 mol l−1sucrose solution for 10 min followed by a 0.2 mol l−sucrose solution for an additional 10 min. The embryos were then washed in Quinn’s Advantage Medium with Hepes (CooperSurgical) plus 5% serum protein substitute (CooperSurgical) and each transferred to a 25 µl microdrop of either Quinn’s advantage cleavage medium (CooperSurgical) or Quinn’s advantage cleavage medium (CooperSurgical) supplemented with 10% serum protein substitute under mineral oil (Sigma, St Louis, MO). The embryos were cultured at 37 °C with 6% CO2, 5% O2 and 89% N2 under standard human embryo culture conditions in accordance with current clinical IVF practice. Embryos used in this study were days post fertilization (DPF) 5–6.
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