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).