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Hepes

Manufactured by CooperSurgical
Sourced in United States

HEPES is a chemical buffer often used in cell culture and biological research applications. It is a zwitterionic organic chemical compound that helps maintain a stable pH environment in various experimental setups.

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3 protocols using hepes

1

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.
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2

Single-Cell Isolation Protocol for Cleavage-Stage Embryos

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The ZP was removed from each embryo by exposure to acidified Tyrode's solution (EMD Millipore) and washed with Ca2+- and Mg2+-free phosphate buffered saline (PBS). Cleavage-stage embryos were disaggregated into single cells, polar bodies, and cellular fragments if present with Quinn's advantage Ca2+- and Mg2+-free medium with HEPES plus 10% human albumin (CooperSurgical) and 0.05% trypsin-EDTA (Thermo Fisher Scientific) as necessary. Each blastomere, polar body, and cellular fragment was washed with Ca2+- and Mg2+-free PBS and collected individually for transfer to a sterile UltraFlux PCR tube (VWR). All of the above was performed under a stereomicroscope equipped with a digital camera (Leica Microsystems), which has movie-making capabilities, to document the collection of every sample. Samples were put into tubes, flash frozen on dry ice, and stored at −80°C. Only embryos for which the disassembly process occurred effectively with no apparent loss of material were carried forward for library preparation and sequencing.
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3

Mouse Oocyte Collection and Culturing

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Mice were maintained in accordance with the policies of the Konkuk University Institutional Animal Care and Use Committee (IACUC). This study was approved by the Konkuk University IACUC (approval number KU12081). Four-week-old ICR mice were purchased from Orient-Bio (Gyeonggi-do, Korea) and were housed in a controlled barrier facility at Konkuk University. The mouse room is equipped with an automated light-dark cycle system and mice were fed a standard rodent chow (LabDiet, St. Louis, MO, USA). Mice were superovulated by injecting 7.5 IU PMSG (Sigma-Aldrich, St. Louis, MO, USA) and 7.5 IU hCG at a 48 h intervals. After 13–14 h after hCG injection, mice were sacrificed and oviducts were collected. Each mouse typically ovulated approximately 17–20 cumulus-oocyte complexes (COCs). COCs were collected by oviduct flushing 13–14 h post-hCG injection. Cumulus cells were removed by treating COCs with hyaluronidase (300 µg/ml) for 2 min. MII oocytes were collected, transferred to Quinn’s Advantage medium with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES, Cooper Surgical, Trumbull, CT, USA) containing 20% fetal bovine serum (FBS, Gibco, Life Technologies, Grand Island, NY, USA), and cultured at 37°C.
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