Apollo solution

Proliferation rates of hPGCs and cells derived from mouse submandibular glands were measured with a 5′-ethynyl-2′-deoxyuridine (EdU) Staining Kit (RiboBio) and with Cell Counting Kit-8 (CCK-8; Dojindo, Shanghai, China) respectively, in accordance with the manufacturers’ instructions.
For the EdU assay, cells were incubated with EdU solution in a 24-well plate for 2 h, fixed in 4% paraformaldehyde at room temperature for 30 min, rinsed with PBS, and stained with Apollo solution (RiboBio). Nuclei were stained with Hoechst 33,342. Hoechst-stained cells and EdU-positive cells were counted using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD).
For the CCK-8 assay, cells were incubated with 10 µl CCK-8 solution in a 96-well plate for 1 hr. Absorbance was measured at 450 nm by using a microplate reader (BioTek Instruments, Winooski, VT); the absorbance level at 12 hr served as baseline.

Cell proliferation ability was measured by EdU assay [26 (link)]. The EdU Apollo Imaging Kit (RiboBio) was used in this study. Fifty μM/well EdU was added, and then GC cells were exposed to 4% paraformaldehyde for 0.5 h. Next, cells were incubated with Apollo solution (RiboBio) and diamidine phenylindole (DAPI; RiboBio). Cell pictures were saved by a microscopy (20 × objective lenses).

CCK-8: Absorbance at a wavelength of 450nm was measured one hour later after Cell Counting Kit-8(BeyotimeInst Biotech, China) reagent was added to each well of a 96-well plate. And the absorbances at 24, 48 and 72h post-transfection were also measured using an Multiscan Go (Thermo, USA). Experiments were repeated at least three times in duplicates.
EdU: Incubation with EdU reagent (Ribobio, China) at 48h post-transfection. And then permeabilization buffer was added to cells at 24h post-incubation followed by washing with PBS. After stained with Apollo solution (Ribobio, China) for 30mins, cells were observed using fluorescence microscopy. Experiments were repeated at least three times in duplicates.