IN A NUTSHELL
Fatty muscles: The negative effects of ectopic fat on bone health and physical function in older adults
This HubLE Method describes the protocol for the generation of bone marrow derived mouse adipocytes.
- Fetal calf serum (FCS)
- Phosphate buffered saline (PBS)
- Hank’s Balanced Salt Solution (HBSS)
- Standard aMEM tissue culture medium (10% FCS, Penicillin and Streptomycin).
- Mice (3-6 months old)
- Forceps, scissors and scalpel
- Needles (19G to 25G)
- Small and large Petri dishes
- Insulin (20mM)
- 3-isobutyl-1-methylxanthine (0.5mM)
- Troglitazone (10mM)
- Dexamethasone (0.25mM)
- Indomethacin (0.1mM)
- Dissect long bones from 3 month-old mice and place in sterile ice-cold BPS
- Once under tissue culture conditions, wash dissected femurs and tibias with ice-cold Hank’s Balanced Salt Solution (HBSS)
- Prepare a solution of HBSS/FCS (1:1)
- Flush out bone marrow stromal cells (BMSC) into HBSS/FCS using a 25 G needle
- Obtain a single cell suspension of BMSC using needles of decreasing size (19G ®25G)
- Centrifuge (300g) for 5 minutes and plate BMSC into large petri dish (2 mice/dish) Incubate BMSC in aMEM supplemented with 10% FCS + P/S and insulin (20nM)
- Incubate BMSC in aMEM supplemented with 10% FCS + P/S and insulin (20nM)
- Once confluent, trypsinise and re-plate cell monlayer in tissue culture plates [Tip No.1]
- Prepare adipocyte differentiation medium (standard aMEM containing 0.5mM 3-isobutyl-1-methylxanthine (IBMX), 0.25µM dexamethasone and 0.1µM indomethacin). [Tip No.2]
- Incubate confluentcells in a adipocyte differentiation medium for up to 21 days.
- Refresh medium every 48 hours
- Identify mature adipocytes by staining for Nile Red ( 1) [Tip No.3]
- We recommend seeding BMSC at 5 x 103cells/cm2in adipocyte differentiation medium (preferably in a 24-well tissue culture plate).
- The differentiation of mature adipocytes can be enhanced by the addition of 1mM Troglitazone to the adipocyte differentiation medium.
- Mature adipocytes can also be visualised by Alizarin Red staining.
- De Ugarte DA, Morizono K, Elbarbary A, et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs. 2003; 174(3): 101-9.
- Idris AI, Sophocleous A, Landao-Bassonga E, et al. Cannabinoid receptor type 1 protects against age-related osteoporosis by regulating osteoblast and adipocyte differentiation in marrow stromal cells. Cell Metab. 2009; 10(2): 139-47.
- To cite this article: B. Li. AI. Idris. Generation of murine adipocytes. HubLE Methods. 1. DOI: 10.13140/RG.2.2.15168.17926
This protocol is for research use only and is not intended for use in human.
David Scott, PhD
NHMRC Career Development Fellow at Monash University, Melbourne, Australia