Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

Published: April 30, 2023

Abstract

Source: Cen, W., et al. A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro. J. Vis. Exp. (2022)

In this video, we describe an in vitro osteoclastic resorption assay on calcium phosphate-coated cell culture plates cultured with human peripheral blood mononuclear cell-derived osteoclasts. The osteoclasts form a characteristic actin ring on the calcium phosphate-coated matrix of the cell culture plate and degrade the matrix within the actin ring region to release calcium and phosphate ions, resulting in the formation of distinct resorption pits within the degraded matrix.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Expansion of OC progenitors Resuspend PBMCs in complete α-MEM (10% FBS, 1% Pen/Strep, 1% amphotericin B) containing 20 ng/mL M-CSF. Seed PBMCs at a density of 2.5 x 105 cells/cm2. Usually, cells isolated from 15-20 mL fresh blood can…

Divulgazioni

The authors have nothing to disclose.

Materials

α-MEM Gibco 32561-029 MEM alpha, GlutaMAX, no nucleosides
amphotericin B Biochrom 03-028-1B Amphotericin B Solution
Calcein Sigma-Aldrich C0875 Calcein
FBS Sigma-Aldrich F7524 fetal bovine serum
Fixation buffer Biolegend 420801 Paraformaldehyde
Hoechst 33342 Promokine PK-CA707-40046 Hoechst 33342
M-CSF PeproTech 300-25 Recombinant Human M-CSF
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)
Triton X-100 Sigma-Aldrich T8787 Alkyl Phenyl Polyethylene Glycol
TrypLE Express Gibco 12605010 Recombinant cell-dissociation enzymes

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Citazione di questo articolo
Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates. J. Vis. Exp. (Pending Publication), e21160, doi: (2023).

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