Mab4360

To begin with, iPSC colonies were fixed using 5% paraformaldehyde and permeabilized with 0.1% Triton X-100. Primary phycoerythrin (PE)-conjugated anti-SSEA-4 antibody (FAB1435P; R&D Systems, Minneapolis, MN) was used for SSEA-4 staining. For TRA-1–60 and TRA-1–81, pretreated colonies were incubated with primary mouse antihuman TRA-1–60 (MAB4360; Merck Millipore, Darmstadt, Germany) and mouse antihuman TRA-1–81 (MAB4381; Merck Millipore). This is followed by incubation with secondary Alexa Fluor 488-conjugated goat antimouse antibody (A11029; Molecular Probes-Invitrogen, Waltham, MA). 4′,6-diamidino-2-phenylindol (Roche Diagnostics, Basel, Switzerland) were used to counterstain cell nuclei.

SEES cells cultured in appropriate conditions were stained for 30 min at 4 °C with primary antibodies and immunofluorescence secondary antibodies. Cells were analyzed with a Cytomics FC 500 Cytometer (Beckman Coulter), and data were analyzed with FC 500 CXP Software ver. 2.0 (Beckman Coulter). Antibodies against human SSEA-1 (R&D Systems; #FAB2155C), SSEA4 (R&D Systems; #FAB1435F), TRA1-60 (Merck Millipore; #MAB4360), and TRA1-81 (Merck Millipore; #MAB4381) were used as primary antibodies in hESCs. PE-conjugated anti-mouse IgG antibodies (BD Pharmingen; #555578) and PE-conjugated anti-mouse IgM antibodies (BD Pharmingen; #553472) were used as secondary antibodies. X-Mean, the sum of intensity divided by the total cell number, was automatically calculated and was utilized for our evaluation.

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 ].

hESCs were passaged on Matrigel-coated plates, and cells were cultured in TeSR-E8 medium. Cells were fixed in 4% paraformaldehyde, and permeabilization and blocking were performed in 5% BSA and 1% Triton X-100 in PBS for 30 min. Cells were stained with anti-NANOG (Abcam, #AB80892), SOX2 (EMD Millipore, #AB5603), TRA-1-60 (EMD Millipore, #MAB4360), TRA-1-81 (EMD Millipore, #MAB4381), SSEA-4 (EMD Millipore, #MAB4304), or OCT4 (Santa Cruz Biotechnology, #SC-5279), all at 1:100. Secondary antibody was applied for 1 hr at room temperature. Nuclei were stained with DAPI (Life Technologies). Images were acquired using a Zeiss IX71 microscope and MetaMorph Advanced software.