To mimic embryonic development in a three-dimensional manner, we used a hanging drop method to differentiate ESCs into EBs. Briefly, ESC colonies were dissociated into single cells and suspended in ESC medium without LIF. To induce formation of EBs, 1000 cells were plated in a 20 µL drop hanging on the lid of culture dish and cultured for 3 days. EBs were collected at day 3 and transferred to ultra-low attachment 6-well plate (Corning) containing fresh ESC medium without LIF. For further suspension culture, medium was refreshed at day 5. EBs were harvested at day 3, 5, and 7 for analysis. For Western blot analysis, total proteins were prepared from ESCs and EBs at day 3, 5, and 7 to detect expressions of HO-1, Oct4, brachyury (R&D, AF2085), SM α-actin (Sigma, A5228), SM22α (Abcam, ab155272), or Smad2 (Cell signaling, #3102). The blots were subsequently probed with α-tubulin antibody (GeneTex, GTX112141) to verify loading. To assess Erk1/2 phosphorylation, blots were probed with a rabbit anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #9101) and α-tubulin antibody for loading.
Af2085
Manufactured by Merck Group
The AF2085 is a specialized laboratory equipment designed for essential research and analytical tasks. It is a versatile instrument that provides reliable and precise measurements. The core function of the AF2085 is to facilitate accurate data collection and analysis, supporting various scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Gtx112141, by GeneTex (1 mentions)
Ultra low attachment 6 well plate, by Corning (1 mentions)
Rabbit anti phospho erk1 2 antibody, by Cell Signaling Technology (1 mentions)
C6219, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Fluorescence conjugated secondary antibody, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
3 protocols using af2085
1
Embryoid Body Formation from ESCs
2
Immunofluorescence Staining of Stem Cells
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Cells were fixed with 4% PFA for 15 min, RT, washed with PBS three times, then permeabilized with 0.3% PBSTr (Triton-X100) for 15 min. Permeabilization is not required for cell surface antigens. After blocking in 3% BSA for 30 min, the cells were incubated with primary antibodies at 4 °C, overnight. The next day, after washing with 0.1% PBST (Tween) three times, the cells were incubated with fluorescence-conjugated secondary antibodies (Abcam, 1:200) for 1 h, RT, then washed with 0.1% PBST three times. The nuclei were stained with DAPI (Sigma-Aldrich, 1:10000). Images were captured with fluorescence microscope (Leica, Germany). The primary antibodies used were: anti-SSEA-4 (Santa Cruz, sc59368, 1:200), anti-OCT4 (Abcam, ab181557, 1:200), anti-TBXT (R&D systems, AF2085, 1:200), anti-CX43 (Sigma-Aldrich, C6219, 1:400), and anti-NR2F2 (Santa Cruz, sc393481, 1:100).
3
Western Blot Analysis of Brachyury and β-Actin
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For immunoblot analyses, cells were lysed with RIPA buffer (150 mM NaCl, 1% Triton X, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0)). Proteins were separated by SDS‐polyacrylamide gel electrophoresis and transferred on nitrocellulose membrane. The membrane was blocked using 5% milk powder in wash buffer (8.5 mM Tris‐HCl, 1.7 mM Tris‐Base, 50 mM NaCl, 0.1% Tween 20 in a. bidest) for 1 h and incubated with primary antibodies against Brachyury (T) (R&D Systems, AF2085) and β-Actin (Sigma Aldrich, A1978) for 2 h at RT. Secondary horseradish peroxidase‐conjugated antibodies were used to detect primary antibodies. Antibodies were diluted in blocking solution. To visualize bound antibodies, Luminol solution was used.
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