Mouse anti human ssea4

AP staining was performed using an Alkaline Phosphatase kit (Millipore, USA). For immunofluorescence, cells were fixed with PBS containing 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, the cells were treated with 0.1% Triton X-100 for 15 min at room temperature before blocking for 2 h with 1 mL of 1× PBS containing 5% bovine serum albumin (BSA). Primary antibodies included mouse anti-human NANOG (1:200, Novus), mouse anti-human OCT4 (1:200, Novus), rabbit anti-human SOX2 (1:50, Novus), mouse anti-human SSEA4 (1:500, Cell Signaling Technology, Beverly, MA, USA), mouse anti-human TRA-1-60 (1:50, Novus), mouse anti-human TRA-1-81 (1:50, Novus), rabbit anti-human VCAM-1 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human STRO-1 (1:50, Novus). The secondary antibodies used were DyLight 488-TFP ester-conjugated goat anti-rabbit IgG antibody (1:100, EarthOx), fluorescein isothiocyanate-conjugated rabbit anti-mouse IgM antibody (1:200, eBioscience), fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody (1:100, Santa Cruz Biotechnology). Nuclei were stained with 1 μg/mL DAPI (Cell Signaling).

To determine the effect of PGE₂ treatment on hTSC stemness by FACS analysis, hTSCs (1.5×10⁶ in 50 µl PBS) were incubated with 20 µl of the appropriate serum in a centrifuge tube at 4°C for 30 min. Subsequently, 0.4 µg of mouse anti-human SSEA-4 (Cell Signaling, Cat. #4755S) or mouse anti-human Stro-1 (Millipore, Cat. #MAB4315) primary antibody was added and incubated at 4°C overnight. The cells were then washed three times with 2% FBS-PBS, followed by centrifugation at 500 g for 5 min/each time. Then the cells were treated with 1 µg Cy3 conjugated goat anti-mouse IgG secondary antibody at room temperature for 2 hrs. The cells treated with the second antibody only were used as a staining negative control. Finally, the cells were washed twice with PBS and fixed in 1% paraformaldehyde, followed by FACS analysis on a BD LSR II Flow Cytometer (BD Biosciences).