Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells

Mapping RNA Polymerase II Occupancy in Embryonic Stem Cells

• Human ES Cells
• Growth Conditions
• Immunohistochemical Analysis of Pluripotency Markers
• Teratoma Formation
• Embryoid Bodies (EB)
• Technology and Protocols
• Antibodies
• Chromatin Immunoprecipitations
• Microarray Design

• Download Data
• Data Analysis

For analysis of pluripotency markers, cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature and incubated overnight at 4°C in blocking solution (5 ml Normal Donkey Solution:195 ml PBS + 0.1% Triton-X)(Figure S2).

Figure S2. Analysis of human ES cells for markers of pluripotency. Human embryonic stem cells were analyzed by immunohistochemistry for the characteristic pluripotency markers Oct4 and SSEA-3. For reference, nuclei were stained with DAPI. Our analysis indicated that >90% of the cells were positive for Oct4 and SSEA-3. Alkaline phosphatase activity was also strongly detected in hESCs.

After a brief wash in PBS, cells were then incubated with primary antibodies to Oct-3/4 (Santa Cruz sc-9081), SSEA-3 (MC-631; Solter and Knowles, 1979), SSEA-4 (MC-813-70; Solter and Knowles, 1979), Tra-1-60 (MAB4360; Chemicon International), and Tra-1-81 (MAB4381; Chemicon International) in blocking solution overnight at 4°C. Following incubation with primary antibody, cells were incubated with either rhodamine red or FITC-conjugated secondary antibody (Jackson Labs) for 2-5hrs at 4°C. Nuclei are stained with 4’,6-diamidine-2-phenylidole dihydrochloride (DAPI). Epifluorescent images were obtained using a fluorescent microscope (Nikon TE300). Data is shown for Oct4 and SSEA-3. Our analysis indicated that >90% of the H9 cells were strongly positive for all pluripotency markers.

Alkaline phosphatase activity of human ES cells was analyzed using the Vector Red Alkaline Phosphatase Subtrate Kit (Cat. No. SK-5100; Vector Laboratories) according to manufacturer’s specifications and the reaction product was visualized using fluorescent microscopy.