Embryomax ksom

Manufactured by Merck Group

EmbryoMax KSOM is a laboratory media solution designed for the culture of mammalian embryos. It provides a balanced set of salts, amino acids, energy substrates, and other components to support the in vitro development of embryos.

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EmbryoMax® Advanced KSOM Embryo Medium EmbryoMax KSOM Embryo Culture EmbryoMax KSOM medium EmbryoMax KSOM medium + amino acids EmbryoMax ® KSOM Mouse Embryo Media EmbryoMax® KSOM + AA with D-glucose and phenol red EmbryoMax KSOM Medium (1X) EmbryoMax

5 protocols using embryomax ksom

Embryo Development Modulation

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Eight‐week‐old C57BL/6J female mice were super‐ovulated by intraperitoneal injection of 10 IU pregnant mare serum gonadotropin (PMSG, SanSheng, Ningbo, China) followed by 10 IU human chorionic gonadotropin (hCG, ShuSheng) 48 h later. Super‐ovulated female mice were caged individually with one fertile male overnight and mating was confirmed by the presence of a vaginal plug. Embryos were obtained by flushing the fallopian tubes and uterine horns with M2 medium (M7167, Sigma–Aldrich). Embryos were cultured in KSOM medium (EmbryoMax KSOM, Sigma–Aldrich, MR‐121), and then 8‐cell stage embryos were treated with DMSO or OSMI‐1, respectively. The developmental potential and polarity of embryos were assessed by immunofluorescence microscopy.

Yu F., Yang S., Ni H., Heng D., Wu X., Yang M., Zhang X., Cao Y., Pei Y., An D., Li D., Liu D., Liu L., Pan L., Chen Q., Zhu X, & Zhou J. (2023). O‐GlcNAcylation Regulates Centrosome Behavior and Cell Polarity to Reduce Pulmonary Fibrosis and Maintain the Epithelial Phenotype. Advanced Science, 10(36), 2303545.

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Real-time Embryo Development Imaging

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Embryos were obtained from naturally mated or superovulated H2B-miRFP720 females mated to either wild-type (CD1) or H2B-miRFP720 males. Embryos were isolated at E1.5 (2-cell), or E2.5 (8-cell) in M2 media and were cultured in Embryomax KSOM (Sigma-Aldrich) under paraffin oil (Life Global Paraffin Oil - LGPO from Cooper Surgical) in a V-shaped imaging chamber at 37°C, with 5% O₂ and 5% CO₂. Images were acquired on an InVi SPIM (Luxendo/Bruker). To limit light exposure to the embryo, we acquired a full 3D image of each embryo at 15- minute intervals, with 2.0 μm z-axis resolution and 0.208 μm x- and y-axis resolution. Typically, the embryos were imaged from the 8-cell stage until the 64-cell stage or to the >100-cell stage, resulting in a duration of 48 hours or more. Raw time-lapse images were compressed to keller-lab-block (klb) format, on the fly.

Nunley H., Shao B., Grover P., Singh J., Joyce B., Kim-Yip R., Kohrman A., Watters A., Gal Z., Kickuth A., Chalifoux M., Shvartsman S., Posfai E, & Brown L.M. (2023). A novel ground truth dataset enables robust 3D nuclear instance segmentation in early mouse embryos. bioRxiv.

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iPSC Blastocyst Injection Assay

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Blastocyst injections were performed using (C57/Bl₆×DBA) B₆D₂F₂ host embryos. All injected iPSC lines were derived from crosses of 129Sv/Jae to C57/Bl₆ mice and could be identified by agouti coat color. Embryos were obtained 24 hr (1 cell stage) or 40 hr (2 cell stage) posthuman chorionic gonadotropin (hCG) hormone priming. To obtain tetraploid (4n) blastocysts, electrofusion was performed at ∼44–47 hr post-hCG using a BEX LF-101 or LF-301 cell fusion apparatus (Protech International). Both fused and diploid embryos were cultured in EmbryoMax KSOM (Millipore) or Evolve KSOMaa (Zenith Biotech) until they formed blastocysts (94–98 hr after hCG injection) at which point they were placed in a drop of Evolve w/HEPES KSOMaa (Zenith) medium under mineral oil. A flat tip microinjection pipette with an internal diameter of 16 μm (Origio) was used for iPSC injections. Each blastocyst received 10–12 iPSCs. Shortly after injection, blastocysts were transferred to day 2.5 recipient CD1 females (20 blastocysts per female). Pups, when not born naturally, were recovered at day 19.5 by cesarean section and fostered to lactating Balb/c mothers.

Buganim Y., Markoulaki S., van Wietmarschen N., Hoke H., Wu T., Ganz K., Akhtar-Zaidi B., He Y., Abraham B.J., Porubsky D., Kulenkampff E., Faddah D.A., Shi L., Gao Q., Sarkar S., Cohen M., Goldmann J., Nery J.R., Schultz M.D., Ecker J.R., Xiao A., Young R.A., Lansdorp P.M, & Jaenisch R. (2014). The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection. Cell stem cell, 15(3), 295-309.

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Modulating Embryo Development via miR-451

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A total of 4–10 pl of the miR-451 inhibitor (50 μmol·l^− 1) was injected into the cytoplasm of mouse MII oocytes. An equal volume of NC inhibitor was injected into the control oocytes. Approximately 60 oocytes were injected each time, and each injection experiment was repeated at least thrice. After injection, the oocytes were introduced into M2 medium for 8 h and then used for IVF. The oocytes injected with miR-451 inhibitor or NC inhibitor were placed in 500 μl EmbryoMax Human Tubal Fluid (Millipore, Billerica, MA, USA) medium under mineral oil. After preincubation of fresh sperm, 100 μl of the sperm suspension (final concentration: 10,000–20,000 spermatozoa·ml^− 1) was added to the drop containing oocytes. The fertilization dishes were incubated at 37 °C in 5% CO₂ and 95% humidified air for at least 5 h. The inseminated oocytes were then cultured in EmbryoMaxKSOM (Millipore) medium. The 2-cell formation rate and blastocyst rate were recorded at days 2 and 4 post-fertilization.

Li X., Zhang W., Fu J., Xu Y., Gu R., Qu R., Li L., Sun Y, & Sun X. (2019). MicroRNA-451 is downregulated in the follicular fluid of women with endometriosis and influences mouse and human embryonic potential. Reproductive Biology and Endocrinology : RB&E, 17, 96.

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Comprehensive Cell Death Evaluation in Mice

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TRIzol reagent was purchased from Invitrogen. ReverTra Ace qPCR RT Master Mix with gDNA Remover Kit and SYBR Green Master Mix were obtained from TOYOBO (Osaka, Japan). Hyaluronidase, Oil Red O, pentobarbital natrium and paraformaldehyde were purchased from Sigma-Aldrich. 3,3′-diaminobenzidine and hematoxylin and eosin (HE) were obtained from Bosterbio. Optimal cutting temperature compound (OCT) was purchased from Sakura Finetek (Torrance, CA, USA). Pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) were obtained from Ningbo Secondary Hormone Corp. (Ningbo, China) . In Situ Cell Death Detection Kit (Fluorescein) was purchased from Roche Applied Sciences. Chemiluminescent HRP Substrate, Embryo max HTF, EmbryoMax M2 medium, Embryo Max KSOM were obtained from Millipore. Oil for embryo culture was purchased from Irvine Science (Santa Ana, CA, USA). Halt Protease & Phosphatase Inhibitor Cocktail was obtained from Pierce. Bicinchoninic acid (BCA) protein assay kit and RIPA lysis and extraction buffer were purchased from Dingguo (Beijing, China). The details, suppliers and dilution of antibodies used in this study are reported in Table 1. All other chemicals were of reagent grade and were obtained from standard commercial sources.

Yang L., Chen L., Lu X., Tan A., Chen Y., Li Y., Peng X., Yuan S., Cai D, & Yu Y. (2018). Peri-ovarian adipose tissue contributes to intraovarian control during folliculogenesis in mice. Reproduction (Cambridge, England), 156(2).

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