Maintenance and Differentiation of 3T3-L1 Preadipocytes

Maintenance and differentiation of 3T3-L1 preadipocytes (provided by Dr. Howard Green) (23 (link)) were carried out as described previously (24 (link)). Deferoxamine (DFO) (Sigma-Aldrich, D-9533) was freshly diluted in Dulbecco's modified Eagle's medium (DMEM), and added to the culture medium at the indicated concentrations. Stock solutions of PIK-III (Selleck Biotech, S7683), bafilomycin A1 (Cayman Chemical Company, 11038), and 2,2’-bipyridyl (FUJIFILM Wako, 042–04241) were prepared by diluting them in dimethyl sulfide (DMSO) at 10, 1 and 100 mM, respectively and added to the medium at the indicated concentrations, where final DMSO concentrations were 0.01%, 0.01% and 0.02%, respectively. Oil red-O (ORO) staining was performed as described previously (25 (link)), and photographed with a GT-X980 scanner (EPSON) and an inverted microscope Primovert (Zeiss). The cell number counting assay was performed at the indicated time points during adipocyte differentiation by detaching cells using 0.25% trypsin (FUJIFILM Wako, 200-13953), 0.75 mg/ml collagenase (FUJIFILM Wako, 17105-041), and 0.5 mg/ml Dispase II (Thermo Fisher, 17105-041) in DMEM.

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Suzuki T., Komatsu T., Shibata H., Tanioka A., Vargas D., Kawabata-Iwakawa R., Miura F., Masuda S., Hayashi M., Tanimura-Inagaki K., Morita S., Kohmaru J., Adachi K., Tobo M., Obinata H., Hirayama T., Kimura H., Sakai J., Nagasawa H., Itabashi H., Hatada I., Ito T, & Inagaki T. (2023). Crucial role of iron in epigenetic rewriting during adipocyte differentiation mediated by JMJD1A and TET2 activity. Nucleic Acids Research, 51(12), 6120-6142.

Publication 2023

Deferoxamine (DFO) bafilomycin A1 GT-X980 scanner Primovert trypsin collagenase Dispase II

3t3 l1 Adipocyte Assay Bafilomycin a1 Bipyridyl Cayman Cells differentiation Collagenase Deferoxamine Dimethyl sulfide Dispase ii Microscope Oil red o Trypsin

Corresponding Organization :

Other organizations : Gunma University, Kyushu University, Nippon Medical School, Gifu Pharmaceutical University, Kanagawa Institute of Technology, Tokyo Institute of Technology, The University of Tokyo, Tohoku University

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Variable analysis

independent variables

Deferoxamine (DFO) concentration
PIK-III concentration
Bafilomycin A1 concentration
2,2'-bipyridyl concentration

dependent variables

Adipocyte differentiation (measured by Oil red-O staining)
Cell number (measured by cell counting assay)

control variables

Maintenance and differentiation of 3T3-L1 preadipocytes (provided by Dr. Howard Green)
Dilution of compounds in Dulbecco's modified Eagle's medium (DMEM)
Final DMSO concentrations for compounds (0.01%, 0.01%, and 0.02%)

controls

No explicit positive or negative controls mentioned

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