Journal: Scientific Reports
Article Title: Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro
Figure Lengend Snippet: Culture of mouse ES on fibroblasts fixed by methanol. J1 mES cells were cultured on MEF cells fixed by MT (MT-MEF) and GA (GA-MEF), respectively. ( A ) Morphology and AP staining of J1 mES cells. ( B ) Growth curve of J1 cells. ( C ) qRT-PCR analysis of Oct4 , Nanog , and Sox2 expressions in J1 cells. ( D ) Flow cytometry analysis of SSEA-1 expression in J1 cells. ( E ) Scanning electron microscope analysis of MT-MEF, GA-MEF, and MMC-MEF. ( F ) J1 cells were cultured on MT fixed C2C12, PEF, and PK-15 cells. Phase 1, MT fixed cells; Phase 2, morphology of J1 cells cultured on MT fixed cells. Scale bar, 200 μm. Data indicate mean ± SD, *P
Article Snippet: After blocking in BSA-blotting buffer (1% BSA and 0.1% Tween 20 in PBS) for 30 min, cells were incubated in BSA-blotting buffer with primary antibodies, including anti-Oct4 (1:200, sc-5279, Santa Cruz), anti-Sox2 (1:200, sc-365823, Santa Cruz), anti-Nanog (1:200, ab80892, Abcam), anti-SSEA-1 (1:200, 4744 S, Cell Signaling Technology), anti-Fibronectin (1:100, 15613-1-AP, Proteintech) and anti-Collagen-IV (1:50, 19797-1-AP, Proteintech), in a humidified chamber at 4 °C overnight.
Techniques: Cell Culture, Staining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Expressing, Microscopy