Fatty muscles: The negative effects of ectopic fat on bone health and physical function in older adults

Introduction

This HubLE Method describes the protocol for the generation of bone marrow derived mouse adipocytes.

Materials

• 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)

Methods [Update]

1. Dissect long bones from 3 month-old mice and place in sterile ice-cold BPS
2. Once under tissue culture conditions, wash dissected femurs and tibias with ice-cold Hank’s Balanced Salt Solution (HBSS)
3. Prepare a solution of HBSS/FCS (1:1)
4. Flush out bone marrow stromal cells (BMSC) into HBSS/FCS using a 25 G needle
5. Obtain a single cell suspension of BMSC using needles of decreasing size (19G ®25G)
6. 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)
7. Incubate BMSC in aMEM supplemented with 10% FCS + P/S and insulin (20nM)
8. Once confluent, trypsinise and re-plate cell monlayer in tissue culture plates [Tip No.1]
9. Prepare adipocyte differentiation medium (standard aMEM containing 0.5mM 3-isobutyl-1-methylxanthine (IBMX), 0.25µM dexamethasone and 0.1µM indomethacin). [Tip No.2]
10. Incubate confluentcells in a adipocyte differentiation medium for up to 21 days.
11. Refresh medium every 48 hours

• Identify mature adipocytes by staining for Nile Red ( 1) [Tip No.3]

Tips [Update]

1. We recommend seeding BMSC at 5 x 10³cells/cm²in adipocyte differentiation medium (preferably in a 24-well tissue culture plate).
2. The differentiation of mature adipocytes can be enhanced by the addition of 1mM Troglitazone to the adipocyte differentiation medium.
3. Mature adipocytes can also be visualised by Alizarin Red staining.

References [Update]

1. 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.
2. 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.

Footnote:

• 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.

02 – HubLE – In a Nutshell – D. Scott