Ab16287

After being seeding on 6-well plates, 253G1 cells seeded onto matrix-coated 6-well plate were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at RT. Then, cells were permeabilized with 0.1% Triton X-100 in PBS at RT for 5 min and blocked with 1% BSA/PBS solution at RT for 30 min. To detect OCT4 marker protein, cells were treated with anti-OCT4 rabbit polyclonal antibody as the primary antibody (ab19857; Abcam) and subsequently with Alexa Fluor488-conjugated anti-rabbit IgG antibody (ab150077; Abcam). To detect SSEA4, cells were treated with anti-SSEA4 mouse monoclonal antibody (ab16287; Abcam) as the primary antibody and subsequently with Alexa Fluor647-conjugated antimouse IgG antibody (ab150115; Abcam). The nuclei were stained with 4′,6-diamidino-2-phenylindole (Dojindo) in 1% BSA/PBS.

ESCs over 85% confluence were passaged as clumps by utilizing Dissociation Buffer (Cat# RP01007; Nuwacell, Hefei, Anhui, China). The relevant markers, including stage-specific embryonic antigen-4 (SSEA4), TRA-1-60, and TRA-1-81, were detected by flow cytometry. The cells were filtered through a 40-µm cell strainer and centrifuged at 500 g for 5 min at 4°C, with the supernatant removed. Later, cells were resuspended in fluorescence-activated cell sorting (FACS) buffer (cat. no. 00–4222; eBioscience, San Diego, CA, USA) at 2 × 10⁶/mL to prepare single-cell suspension. The cells were incubated with antibodies anti-SSEA4 (1:1000, ab16287, Abcam, Cambridge, UK), anti-TRA-1-60 (10 µg/mL, MAB4360, Merck, Kenilworth, NJ, USA), and anti-TRA-1-81 (0.5 µg/mL, gtx48034, GeneTex, Irvine, CA, USA) on ice for 30 min, respectively. Subsequently, cells were reacted with secondary antibody goat anti-mouse immunoglobulin G (IgG) Alexa Fluor® 488 (1:2000, ab150113, Abcam) for 30 min. The mouse IgG (1 µg/mL, ab170190, Abcam) served as the isotype control. After two washes, cells were resuspended in phosphate-buffered saline (PBS) with 2% FBS and cells with the positive expression of SSEA4, TRA-1-60 and TRA-1-81 were analyzed by a flow cytometer (FACSAria III; BD Biosciences, San Jose, CA, USA). The results were analyzed using FlowJo (version 10.0.7r2; FlowJo LLC, Ashland, OR, USA). [42 ].

Immunofluorescence staining of the Pdot-labeled MSCs was performed in accordance of manufacturers' instructions. In brief, cells were fixed with 4% PFA for 20 mins at room temperature, permeabilized by using 0.5% TtitonX-100 for 20 mins at room temperature. After blocking with 5% BSA for 1 h at 37 °C, cells were incubated with primary antibodies including OCT-4 (ab19857, Abcam), NANOG (ab109250, Abcam), and SSEA-4 (ab16287, Abcam) at 4 °C overnight, followed by incubation with Alexa Fluor^® 488-labeled secondary antibodies for 2 hours at room temperature in dark. Tubulin structure was stained by biotin anti-α-tubulin antibody (627904, Biolegend) and Alexa 488-streptavidin (S11223, Invitrogen).