Adipose tissue-derived stem cells (ASC) were isolated from inguinal subcutaneous WAT. Adipose tissue was minced and washed with phosphate-buffered saline (PBS). The minced cells were trapped on fiber mesh and cultured in ASC growth medium (Mirai Bio Kobo, Tokyo, Japan) at 37°C with 5% CO2 for 10 days. After 10 days, isolated ASC were plated at a density of 2.0×104 cells/cm2 in 6-well plates with 2.0 mL of plating medium (DMEM + 10% FBS). When the cells reached 100% confluence, differentiation was induced by the addition of adipogenic differentiation medium (adipogenic base medium + adipogenic supplement) (R&D Systems). Every 3–4 days, the differentiation medium was removed and replaced. Differentiation was complete after 10 days, at which time adipogenic-induced cells will showed morphological changes and lipid vacuoles. To quantitate or visualize the effect of treatment on lipid accumulation, we performed Oil Red O staining.
Adipogenic supplement
Manufactured by R&D Systems
Adipogenic supplement is a laboratory product designed to support the in vitro differentiation of preadipocytes into mature adipocytes. The supplement contains a proprietary mixture of growth factors, hormones, and other compounds that promote the adipogenic differentiation process.
Automatically generated - may contain errors
Lab products found in correlation
Stemxvivo osteogenic adipogenic base media, by R&D Systems (2 mentions)
Osteogenic supplement, by R&D Systems (2 mentions)
Adipogenic differentiation medium, by R&D Systems (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Roswell park memorial institute (rpmi), by Sartorius (1 mentions)
Lymphoprep, by Progen Biotechnik (1 mentions)
Cfx384 real time system machine, by Bio-Rad (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Direct zol rna isolation kit, by Zymo Research (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Its supplement, by R&D Systems (1 mentions)
6 protocols using adipogenic supplement
1
Adipose Stem Cell Isolation and Differentiation
2
Adipogenic Differentiation of Mesenchymal and Epicardial Stem Cells
3
Adipogenic Differentiation of AMSCs
4
Isolation and Differentiation of Mesenchymal Stromal Cells from Bone Marrow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For cell preparation, mononuclear cells (MNC) were isolated from BM aspirates using density gradient centrifugation with LymphoPrep™ (PROGEN Biotechnik, cat. #1,114,547, Heidelberg, Germany).
To expand the mesenchymal stromal cell fraction, isolated MNCs were cultured in the Mesencult medium [Mesencult™ Proliferation Kit (human), STEMCELL Technologies Inc, cat. #05411, Vancouver, BC, Canada], with an addition of 10% AB human serum and 1% penicillin–streptomycin solution. BM stromal cells were generated up to passage (P) 3. To enrich and expand the MSC fraction, the isolated MNCs were cultured in the starvation medium RPMI (Biological Industries, cat. #1640, Beit-Haemek, Israel), with an addition of 10% FBS/human serum and 1% penicillin–streptomycin solution.
To induce MSC differentiation into adipocytes/osteocytes, BM-MSCs were stimulated using adipogenic or osteogenic differentiation inducing factors. In brief, P0 MSCs were cultured for 21 days at 370C in either adipogenic or osteogenic medium (basal medium: MEM-α Biological Industries, cat. #01–042-1A; adipogenic supplement: R&D Systems, cat. #CCM011, Minneapolis, MN, USA; osteogenic supplement: R&D Systems, cat. #CCM008). The obtained cells are further referred to as “induced” MSCs, adipocytes or osteocytes. The MSCs that have not undergone such stimulation are further termed “non-induced”.
To expand the mesenchymal stromal cell fraction, isolated MNCs were cultured in the Mesencult medium [Mesencult™ Proliferation Kit (human), STEMCELL Technologies Inc, cat. #05411, Vancouver, BC, Canada], with an addition of 10% AB human serum and 1% penicillin–streptomycin solution. BM stromal cells were generated up to passage (P) 3. To enrich and expand the MSC fraction, the isolated MNCs were cultured in the starvation medium RPMI (Biological Industries, cat. #1640, Beit-Haemek, Israel), with an addition of 10% FBS/human serum and 1% penicillin–streptomycin solution.
To induce MSC differentiation into adipocytes/osteocytes, BM-MSCs were stimulated using adipogenic or osteogenic differentiation inducing factors. In brief, P0 MSCs were cultured for 21 days at 370C in either adipogenic or osteogenic medium (basal medium: MEM-α Biological Industries, cat. #01–042-1A; adipogenic supplement: R&D Systems, cat. #CCM011, Minneapolis, MN, USA; osteogenic supplement: R&D Systems, cat. #CCM008). The obtained cells are further referred to as “induced” MSCs, adipocytes or osteocytes. The MSCs that have not undergone such stimulation are further termed “non-induced”.
5
Adipogenesis Regulation by ZNF467 and PCBP3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AMSCs were plated in six-well plates (day -1) and transfected with siRNAs against ZNF467 and PCBP3 as described above. Two days after siRNA transfection (day 1) adipogenesis was induced by addition of adipogenic supplement (R&D Systems, Minneapolis, MN) to maintenance medium. Media were changed and differentiation cocktail replenished every 2 days. 9. mRNA quantitative real-time reverse transcriptase PCR (RT-qPCR) mRNA was isolated using Direct-zol RNA isolation kit (Zymo Research) as described above. Isolated RNA was reverse transcribed into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Gene expression was quantified using realtime PCR. Each qPCR reaction was performed with 10 ng cDNA per 10 μL, QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) and the CFX384 real-time System machine (Bio-Rad, Hercules, CA). Transcript levels were quantified using 2 ΔΔCt method and normalized to the housekeeping gene GAPDH (set at 100). Gene-specific primer sequences are shown in Table 1.
6
Differentiation of GBM CD105+ Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GBM CD105+ cells at low passage (P3-P5) were used for mesenchymal stem cell (MSC) differentiation assays using a human MSC identification kit (R&D) according to the protocol provided by the manufacturer. Briefly, GBM CD105+ cells were cultured in human/mouse StemXVivo Osteogenic/ Adipogenic Base Media (R&D) with Adipogenic Supplement(R&D) and Osteogenic Supplement(R&D), respectively, for up to 14 days for adipogenic differentiation or osteogenic differentiation. For chondrogenic differentiation, cells were culture in human StemXVivo Chondrogenic Media (R&D) supplemented with ITS Supplement (R&D) and pelleted in 5 ml tube for up to 20 days. Cells were fixed and detected by anti-hFABP4, anti-hOsteocalcin and anti-hAggrecan immunohistochemistry.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!