The eggs were then washed through dye-free medium and placed in 50 μM FluoZin-3 in hCZB as described above.
Gametes were then washed through dye-free medium and placed in 50 μM FluoZin-3 in Ca-free hCZB as described above.
In brief, the TESs were cut into small disks (25 mm in diameter and 5 mg in weight), fixed by a cell-culture ring designed by Zhu et al.41, sterilized by electron-beam irradiation using a linear accelerator (Precise, Elekta, Crawley, U.K). with a total dose of 100 cGy, soaked in the cell culture medium, and placed in a 12-well TCP.
A sterile inoculating loop was used to collect a single 24-h-old colony of isolate RH5 culture from LB agar medium and placed on a slide.
The bands of glycolipids were measured, absorbance (410 nm), using a densitometer and quantified by applying a standard curve of five concentrations of hexenyl Gb3. 5 g of CELL YARD™ beads (Pentax Co). were equilibrated in 100 ml medium and placed in a spinner flask (Shibata scientific technology ltd).
Both fractions were resuspended in medium and placed in chambers slides at a concentration of 1×106 cells per mL.
Separated blastomeres were washed 4 times in pre-equilibrated CCM medium and placed separately in a lysis buffer for the RNA extraction assay.
The coverglass with attached cells was rinsed, and the chambers filled with CO2-independent medium and placed on the microscope stage.
The remaining 16×106 cells were pelleted and resuspended in the staining medium and placed at 37°C for 5 minutes.
EP medium is an isotonic OET-compatible buffer of minimal electric conductivity (10 mS/m).. Embryo cohorts were collected in a blinded fashion, suspended in 50 100 µL of EP medium, and placed onto the OET embryo sorting platform.
Following incubation, Ears were removed from medium and placed individually into medicons (Becton Dickinson, Franklin Lakes, New Jersey) with 500 µl of their own medium and run in a Medimachine™ (Becton Dickinson) for 5 minutes.
