Celltracker red cmfda

1 × 10⁶ cells/mL of either Hep3B or Thle-3 were seeded on sterile coverslips (25-mm, No. 1.5) coated with 0.01% poly-l-lysine and allowed to adhere for 4–24 h at 37 °C. 10 μg (~2.4 × 10¹² particles) of either WT or AF-20 VLPs were incubated with the cells for 2 h at 37 °C, washed three times with 1× PBS, fixed with 3.7% formaldehyde (10 min at RT), and mounted with SlowFade Gold. Prior to fixation, cells were stained with CellTracker Red CMFDA (Invitrogen) to visualize cytoplasm and Hoechst 33342 (Invitrogen) to visualize the nucleus. Three-color images were acquired using a Zeiss LSM510 META (Carl Zeiss MicroImaging, Inc.) operated in Channel mode of the LSM510 software; a 63×, 1.4-NA oil immersion objective was employed in all imaging. Typical laser power settings were: 30% transmission for the 405-nm diode laser, 5% transmission (60% output) for the 488-nm Argon laser, 100% transmission for the 543-nm HeNe laser, and 85% transmission for the 633-nm HeNe laser. Gain and offset were adjusted for each channel to avoid saturation and were typically maintained at 500–700 and −0.1, respectively. 8-bit z-stacks with 1024 × 1024 resolutions were acquired with a 0.7 to 0.9-μm optical slice. LSM510 software was used to overlay channels and to create 3D projections of z-stack images.

The collected zygotes and blastocysts were stained with fluorescent dye- Cell Tracker Red CMFDA (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction, and then transferred into pseudopregnant female recipients. The migration of embryos in the fallopian tubes and uterus was examined under fluorescence microscopy and recorded the location of each embryo in uterine horns from 18:00 on day 3 to 8:00 on day 4.