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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

Abstract

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

Introduction

Osteoclasts (OCs) are tissue-specific macrophages derived from hematopoietic stem cells (HSCs), playing a pivotal role in bone remodeling together with osteoblasts1. Sex hormone-induced, immunological, and malignant bone disorders that destroy bone systemically or locally are due to excess osteoclastic activity, including menopause-related osteoporosis2, rheumatoid arthritis3, periodontal disease4, myeloma bone disease5, and osteolytic bone metastasis6. In contrast, defects in OC formation and function can also cause osteopetr....

Protocol

The protocol was reviewed and approved by the local ethics committee (approval number 287/2020B02).

1. Preparation of calcium phosphate coated cell culture plates

  1. Preparation of calcium stock solution (25 mM CaCl2·2H2O, 1.37 M NaCl, 15 mM MgCl2·6H2O in Tris buffer)
    1. Prepare 50 mM Tris buffer and adjust pH to 7.4 using 1 M HCl.
    2. Set up a glass beaker on a magnetic stirrer and add 100 mL of 50.......

Representative Results

The calcium phosphate coating on the bottom of cell culture plates was performed in two coating steps comprising a 3-day pre-calcification and a 1-day calcification step. As shown in Figure 1, uniformly distributed calcium phosphate was obtained on the bottom of the 96-well plates. The coating adhered very well to the bottom after the performed washing steps.

Discussion

Here we describe a simple and reliable method for an osteoclastic resorption assay using OCs derived and expanded in vitro from PBMCs. The used CaP coated cell culture plates can be easily prepared and visualized using lab-available materials. In addition to unsorted PBMCs adopted in this protocol, OCs generated from murine monocytic cells21 and bone marrow macrophage cells17 have also been cultured on similar synthetic substrates for pit assay, thus these cell sou.......

Acknowledgements

This work was partially funded by the China Scholarship Council [CSC No. 201808440394]. W.C. was financed by CSC.

....

Materials

NameCompanyCatalog NumberComments
AgNO3SERVA Electrophoresis GmbH35110Silver nitrate
a-MEMGibco32561-029MEM alpha, GlutaMAX, no nucleosides
amphotericin BBiochrom03-028-1BAmphotericin B Solution
CaCl2Sigma-Aldrich21097-50GCalcium chloride Dihydrate
CalceinSigma-AldrichC0875Calcein
FBSSigma-AldrichF7524fetal bovine serum
FicollCytiva17144002Ficoll Paque Plus
Fixation bufferBiolegend420801Paraformaldehyde
HClMerk1.09057.1000Hydrochloric acid
Hoechst 33342PromokinePK-CA707-40046Hoechst 33342
M-CSFPeproTech300-25Recombinant Human M-CSF
MgCl2Sigma-Aldrich7791-18-6Magnesium chloride
Na2HPO4AppliChem GmbHA2943,0250di- Sodium hydrogen phosphate anhydrous
NaClMerkS7653-250GSodium chloride
NaHCO3MerkK15322429Bicarbonate of Soda
PBSLonza17-512FDulbecco's Phosphate Buffered Saline (1X), DBPS without Calcium and Magnesium
Pen-StrepLonzaDE17-602EPenicillin-Streptomycin Mixture
Phalloidin-Alexa Fluor 546InvitrogenA22283Alexa Fluor 546 Phalloidin
RANKLPeproTech310-01Recombinant Human sRANK Ligand (E.coli derived)
TrisSigma-Aldrich93362Tris(hydroxymethyl)aminomethan
Triton X-100Sigma-AldrichT8787Alkyl Phenyl Polyethylene Glycol
TrypLE ExpressGibco12605010Recombinant cell-dissociation enzymes

References

  1. Jacome-Galarza, C. E., et al. Developmental origin, functional maintenance and genetic rescue of osteoclasts. Nature. 568 (7753), 541-545 (2019).
  2. Moller, A. M. J., et al. Aging and menopause ....

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