Te300

Following IVM, images of denuded metaphase II (MII) oocytes in each experimental group were recorded using a digital camera (DS-L3; Nikon) attached to an inverted microscope (TE300; Nikon). The size of each part of the matured oocyte was measured using ImageJ software (version 1.49q; National Institutes of Health, USA), and sizes of the PVS and cytoplasm were calculated as described previously [22 (link)]. Briefly, mean diameters of oocytes were calculated by averaging the longest and the shortest diameters of each oocyte. The PVS size was calculated by subtracting the diameter of ooplasm from the inner diameter of zona pellucida.

After electrical activation, the PA embryos were exposed to 5 µg/mL CB and SCNT embryos with 0.4 µg/mL demecolcine combined with 1.9 mM 6-dimethylaminopurine in IVC medium for 4 h. The SCNT and PA embryos were subsequently washed three times in fresh IVC medium, transferred into 30-µL IVC droplets under mineral oil, and cultured at 39°C in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ for 7 days. Cleavage and blastocyst formation were evaluated on Days 2 and 7, respectively, with the day of SCNT or PA designated as Day 0. Count of total cell number per blastocyst was conducted using Hoechst 33342 staining under an epifluorescence microscope (TE300; Nikon).

A fast monochromator (Polychrome II; Till Photonics, Inc., Germany) was used as an excitation light source. A fused quartz light guide was used to divert the excitation light to an inverted microscope (TE-300; Nikon, Tokyo, Japan). An oil immersion lens (40x, NA 1.3) was used. A near infrared filter (Chroma Technology Corp., Bellows Falls, VT, USA) was used between the microscope illuminator and specimen to monitor the object field with a charge-coupled device (CCD) camera (FTM1800NH/HGI; Philips, Salt Lake, USA). The image was captured with a BT878-based TV capture board, and the object field or cell area, as a pixel unit, was measured with custom-made software. The object field area was set with a field diaphragm (Nikon, Tokyo, Japan). Four photomultiplier tubes (PMTs) were used to detect emission wavelengths, and each had band-pass filters (450, 500, 590, and 640 nm). Dichroic mirrors and band-pass filters were purchased from Chroma (Brattleboro VT, USA). A photon counting method was applied with the combination of a photomultiplier tube (R2949; Hamamatsu, Hamamatsu Japan), photon counter unit (C3866; Hamamatsu, Hamamatsu, Japan), and high-speed counter (NI 6602; National Instruments, Austin, USA). Custom-made driving software was used to control and sample data. A diagram of the whole system is shown in Fig. 1.

The nude mice used in this study were treated following the experimental animal ethics guidelines issued by the China Medical University. The study was approved by the Institutional Animal Research Committee of China Medical University. Four-week-old female BALB/c nude mice were purchased from Charles River (Beijing, China), and the axilla or tail vein of each mouse was subcutaneously or intravenously inoculated with 5×10⁶ or 2×10⁶ tumor cells, respectively, in 0.2 mL of sterile PBS. Six weeks after inoculation, mice were euthanized and autopsied to examine tumor growth and dissemination. A portion of tissue from the tumor and each organ was fixed in 4% formaldehyde (Sigma) and embedded in paraffin. Serial 4-μm-thick sections were prepared and stained with hematoxylin and eosin (H&E), and the stained sections were examined under a microscope (Nikon TE300, Melville, NY, USA).