To isolate ICMs, we subjected late blastocysts to immunosurgery (Solter and Knowles, 1975 (link)). In brief, in vitro cultured single and double embryos were incubated with anti-mouse whole serum (M5774-2ML, Sigma-Aldrich) and sera complement guinea pig (S1639-5ML, Sigma-Aldrich) diluted to 20% in M2 medium for 45 min each. After elimination of trophectoderm cells by pipetting with a narrow glass capillary in M2 medium, ICMs were seeded in individual hanging drops of pre-warmed IVC-1 medium (Bedzhov et al., 2014 (link)) (M11-25, Cell Guidance Systems) and cultured for 24, 48, and 72 h at 37°C in 5% CO2 (Figure 2 A).
M5774 2ml
Manufactured by Merck Group
M5774-2ML is a laboratory equipment product from Merck Group. It is a 2 mL vial containing a specific chemical compound or reagent. The core function of this product is to provide a standardized and controlled quantity of the material for use in various laboratory procedures and experiments. No further interpretation or extrapolation on the intended use of this product is provided.
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2 protocols using m5774 2ml
1
Isolation and Culture of Inner Cell Masses from Blastocysts
2
Embryo Development and Quality Evaluation
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The activated oocytes were washed three times in PBS (gibco, 21600-051-10x1L) and then cultured in potassium simplex optimization medium supplemented by 10% fetal bovine serum (Sigma, A2153-50G) at 37 o C under 5% CO2 (New Brunswick, Galaxy 170R CO2 incubator, Eppendorf) up to 5 days. The development rate of the embryo was calculated at each stage (2-cell, 4-cell, morula, blastocyst, and hatched blastocyst), by dividing the number of embryos at each stage by total activated oocytes in each treatment group. Embryo quality was calculated based on the amount of blastocyst, inner cell mass (ICM) and trophectoderm cells. Total ICM and trophectoderm cells were determined by exposed blastocyst to rabbit anti-mouse serum antibody (Sigma, M5774-2ML) for 1-hr and complement sera from guinea pig (Sigma, S1639-5ML) for 30 minutes, and followed by staining using Hoechst 34580 (Sigma, 63493-5MG) -Propidium iodide (Sigma, 81845-25MG). ICM cells located on the inside of the embryo were blue, whereas trophectoderm cells on the outside were red (Hine et al., 2008) .
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