Can anyone suggest a suitable protocol for studying adipogenic differentiation with human bone marrow derived mesenchymal stem cells (BM-MSC) - primary culture? I have found many articles but each article details different cultivation procedures.
One suggestion is the StemPro Adipogenesis medium kit from Gibco. It comes with protocol. It's very easy to use. You just need to add the adipogenic serum and antib-antim. And it is ready to use. After cells confluence you starts the induction. Follow the protocol which comes with the product, it also have directions to do the staining protocol after induction.
I use this protocol for BM-MSCs: cells at passage 3 were seeded at a density of 2x104 cells/cm2 in basal medium. After 24 hours, medium was switched to Adipogenic Induction Medium, consisting of DMEM-High glucose, supplemented with 10% FBS,1% L-glut 1% Pen-Strep, 1µM dexamethasone, 1µM indomethacin, 500 µM 3-isobutyl-1-methylxantine (IBMX) and 10 µg/ml human recombinant insulin. Change medium twice a week.
Patrizia Ferretti (UCL Institute of Child Health, London, UK) We differentiate confluent human mesenchymal stem cells from various sources along the adipocytic lineage by incubating them in a medium containing DMEM, 10% ES-FBS, 10 ng/ml insulin, 500 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone, and 1 mM rosiglitazone (Guasti et al., 2012, Stem Cells Transl Med; 1:384-95). It works well for us.
If I were doing this, I would use the same protocol detailed in the paper first describing the various assays for MSCs. The paper is here:http://www.ncbi.nlm.nih.gov/pubmed/10102814 The adipogenic assay was part of this. Since Lonza sells the MSCs, I think they also have a kit for the adipogenic assay. Ah, yes they do. They have an adipogenic differentiation medium:
http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/adult-stem-cells-and-media/human-mesenchymal-stem-cells-media/hmsc-mesenchymal-stem-cell-adipogenic-differentiation-medium.aspx
Good luck.
Hello Guys
I am working on adipogenesis in mouse embryo fibroblast cells (MEF)
in process of differentiation cells come off easily from tissue culture plate
Please suggest me what coating you use on tissue culture plates (specifically for MEF cell lines)
Hi Krishna,
Commonly used coating for MEF cultures is 0.1% Gelatin in distilled water and autoclave after adding Gelatin.
For coating, add around 5-6 ml of Gelatin to 1 T75cm2 plate and incubate for minimum 20-30mins. Then you can proceed to seed your cells.
Good Luck
I had a related question- Does the seeding density have any effect on differentiation of BM-MSCs?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques