A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

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 mM Tris buffer.
   3. Weigh out 0.368 g of CaCl₂·2H₂O, 8.0 g of NaCl, and 0.305 g of MgCl₂·6H₂

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.......

Materials

Name	Company	Catalog Number	Comments
AgNO3	SERVA Electrophoresis GmbH	35110	Silver nitrate
a-MEM	Gibco	32561-029	MEM alpha, GlutaMAX, no nucleosides
amphotericin B	Biochrom	03-028-1B	Amphotericin B Solution
CaCl2	Sigma-Aldrich	21097-50G	Calcium chloride Dihydrate
Calcein	Sigma-Aldrich	C0875	Calcein
FBS	Sigma-Aldrich	F7524	fetal bovine serum
Ficoll	Cytiva	17144002	Ficoll Paque Plus
Fixation buffer	Biolegend	420801	Paraformaldehyde
HCl	Merk	1.09057.1000	Hydrochloric acid
Hoechst 33342	Promokine	PK-CA707-40046	Hoechst 33342
M-CSF	PeproTech	300-25	Recombinant Human M-CSF
MgCl2	Sigma-Aldrich	7791-18-6	Magnesium chloride
Na2HPO4	AppliChem GmbH	A2943,0250	di- Sodium hydrogen phosphate anhydrous
NaCl	Merk	S7653-250G	Sodium chloride
NaHCO3	Merk	K15322429	Bicarbonate of Soda
PBS	Lonza	17-512F	Dulbecco's Phosphate Buffered Saline (1X), DBPS without Calcium and Magnesium
Pen-Strep	Lonza	DE17-602E	Penicillin-Streptomycin Mixture
Phalloidin-Alexa Fluor 546	Invitrogen	A22283	Alexa Fluor 546 Phalloidin
RANKL	PeproTech	310-01	Recombinant Human sRANK Ligand (E.coli derived)
Tris	Sigma-Aldrich	93362	Tris(hydroxymethyl)aminomethan
Triton X-100	Sigma-Aldrich	T8787	Alkyl Phenyl Polyethylene Glycol
TrypLE Express	Gibco	12605010	Recombinant cell-dissociation enzymes