Generating Adipocytes from Mesenchymal Stem Cells

BM cells were flushed out from human fetal femoral bones (Advanced Bioscience Resources), and cultured in DMEM to generate MSCs [46 (link)]. Immunophenotyping of MSCs was analyzed by flow cytometry. To generate mature adipocytes, MSCs were cultured in adipocyte medium (Lonza) for 2 weeks. Adipocytes were also generated from the adipocyte precursor (pre-adipocyte) cell line PCS-210-010 (ATCC), or pre-WAT cells isolated from subcutaneous fat (LONZA), or isolated from the BM aspirates of healthy adults or MM patients, and further purified by 10 min centrifugation at 186 g [47 (link)]. Mature adipocytes were fixed with 4.0% paraformaldehyde, stained with Oil red O for 1 hour, and observed under light microscopy [48 (link)].

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Liu Z., Xu J., He J., Liu H., Lin P., Wan X., Navone N.M., Tong Q., Kwak L.W., Orlowski R.Z, & Yang J. (2015). Mature adipocytes in bone marrow protect myeloma cells against chemotherapy through autophagy activation. Oncotarget, 6(33), 34329-34341.

Publication 2015

PCS-210-010

Adipocytes Adults Bones Cell line Cells Centrifugation Cultured medium Femoral Fetal Flow cytometry Human Light microscopy Paraformaldehyde Patients Subcutaneous fat

Corresponding Organization : The University of Texas MD Anderson Cancer Center

Other organizations : Children's Nutrition Research Center at Baylor College of Medicine

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Protocol cited in 1 other protocol

Variable analysis

independent variables

Cell source for generating mature adipocytes (BM-derived MSCs, adipocyte precursor cell line PCS-210-010, pre-WAT cells isolated from subcutaneous fat, BM aspirates of healthy adults or MM patients)

dependent variables

Adipocyte maturation as observed by Oil red O staining and light microscopy

control variables

Culture conditions for generating mature adipocytes (DMEM and adipocyte medium)
Fixation of mature adipocytes with 4.0% paraformaldehyde

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

To optimize the experimental performance, the Input protocol and Protocol 3 suggest using bone marrow aspirates or adipose tissue samples from healthy adult donors after obtaining informed consent and approval from the Institutional Review Board (Protocol 3).

The Input protocol, Protocol 2, and Protocol 4 mention the use of specific cell surface markers (CD44, CD90, CD166, BODIPY, CD24, Sca-1) and staining techniques (Oil Red O) to validate the identity and purity of mature adipocytes generated from MSCs or pre-adipocyte cell lines.

Protocol 2 and Protocol 4 provide details on the differentiation of MSCs into mature adipocytes, including the use of specific culture media and supplements (e.g., dexamethasone, indomethacin, insulin, 3-isobutyl-L-methylxanthine) for 2 weeks.

The Input protocol, Protocol 1, and Protocol 2 suggest using 4.0% paraformaldehyde fixation and Oil Red O staining to visualize and confirm the presence of mature adipocytes under light microscopy.

Protocol 4 highlights the importance of using appropriate controls, such as antibodies against specific cell surface markers (CD29, CD34, CD45, CD90) to verify the identity of MSCs (ADSC and BMSC) by flow cytometry.

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As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

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