A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Summary

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Abstract

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

Introduction

Bone is an ever-changing organ and is remodeled by osteoblasts and osteoclasts throughout life. Osteoclasts are responsible for mineralized matrix resorption and osteoblasts synthesize and secrete new bone matrices¹. The balance between bone resorption and bone formation is crucial for bone health including maintenance of bone mass and response to stimulation and injury. If this balance is disrupted, a series of bone metabolic diseases may occur, including osteoporosis and periodontal diseases. In these diseases, bone mass loss resulting from osteoclastic bone resorption exceeds the bone forming capacity of osteoblasts²<....

Protocol

All procedures relating to the animals were performed according to the protocol approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC) and were approved by the Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology.

1. Preparation

1. Generate osteoclast specific Raptor deletion mice (Raptor^fl/fl; Ctsk-cre, hereafter Rap^Ctsk) by mating Raptor^fl/fl mice with Ctsk-cre mice. Use the Raptor^fl/fl mice as the WT control in this study.
2. Have a container with ice to ke....

Results

Using the present protocol, a large number of giant osteoclasts were seen on day 6; if giant osteoclasts are not seen, one more day of osteoclast differentiation may be needed (Figure 1). Successful osteoclast formation was confirmed by TRAP staining (Figure 2A). Osteoclasts were giant wine red/purple cells with more than 3 nuclei. More than 250 osteoclasts were obtained in each well of the 96-well plate in WT BMMs (

Discussion

The osteoclastogenic assay is the most widely used method to isolate and culture osteoclasts in vitro^12,13. While several RANKL-based osteoclast inductions have been described^13,14,15, the present study described a protocol with some modifications based on the previous methods.

In the previous study, BMMs were plating immediately afte.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Raptorfl/fl mice	The Jackson Laboratory	013188
Ctsk-cre mice			a gift from S. Kato, University of Tokyo, Tokyo, Japan
α-MEM	Corning	10-022-CVR
Glutamine	Gibico	25030081
Penicillin streptomycin	Gibico	15140122
Fetal calf serum	BioInd	04-001-1A
Recombinant mouse M-CSF protein	R&D	Q3U4F9
Recombinant mouse RANKL protein	R&D	Q3TWY5
RBC lysis buffer	Beyotime	C3702
Trypan blue	Sigma-Aldrich	302643
Acetone	Shanghai Chemical Co. Ltd.
Citrate solution	Sigma-Aldrich	915
Formaldehyde solution	Shanghai Chemical Co. Ltd.
Acid Phosphatase, Leukocyte (TRAP) Kit	Sigma-Aldrich	387A-1KT
Fast Garnet GBC Base solution	Sigma-Aldrich	3872
Sodium Nitrite Solution	Sigma-Aldrich	914
Naphthol AS-BI Phosphate Solution	Sigma-Aldrich	3871
Acetate solution	Sigma-Aldrich	3863
Tartrate solution	Sigma-Aldrich	3873
Dulbecco's phosphate-buffered saline	Corning	21-031-CVR
L-tartaric acid	Sigma-Aldrich	251380
Sodium tartrate dibasic dehydrate	Sigma-Aldrich	s4797
Glycine	Shanghai Chemical Co. Ltd.
MgCl2	Shanghai Chemical Co. Ltd.
ZnCl2	Shanghai Chemical Co. Ltd.
NaOH	Shanghai Chemical Co. Ltd.
Phosphatase substrate	Sigma-Aldrich	P4744
anti-Raptor	Cell Signaling Technology	2280
anti-P-ribosomal protein S6 (S235/236)	Cell Signaling Technology	2317
anti-ribosomal protein S6	Cell Signaling Technology	2211
anti-β-actin	Santa Cruz Biotechnology	sc-130300
37% formaldehyde	Xilong scientific
polyvinylidene fluoride (PVDF) membrane	Bio-Rad
Western Chemiluminescent HRP Substrate (ECL)	Millipore	00000367MSDS
IX71	Olympus
Envision	Perkin Elmer
0.45-mm Syringe
Scissor
Mosquito forcep