In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction
Published: April 30, 2023
Abstract
Source: Kampleitner, C. et al. Biological Compatibility Profile on Biomaterials for Bone Regeneration. J. Vis. Exp. (2018)
In this video, we describe a method for the detection of osteoblast-secreted calcium deposits using an anionic dye, Alizarin Red S. The cultured osteoblasts are first stained and then the dye is recovered for colorimetric quantification of calcium deposits or the degree of osteoblast mineralization.
Protocol
1. Osteoblast mineralization assay Pre-incubate 14 mm diameter β-tricalcium phosphate (β-TCP) disks in 1 mL of bone growth medium (BGM) in a 24-well suspension culture plate at 37 °C and 5% CO₂ for 24 h before adding the osteoblasts (OBs). Use standard tissue culture plates for controls and suspension culture plates for the biomaterial samples to avoid cell attachment to the plastic. Aspirate the pre-incubation medium and then resuspend primary mouse OBs in BGM. Add 8.8 x 10S…
Disclosures
The authors have nothing to disclose.
Materials
| β-tricalcium phosphate disks (β-TCP, 14 mm) | Disks were sintered in a muffle furnace at a temperature of 1150 °C. Samples were UV-irradiated (15 min for each side) before using in cell cultures | ||
| Alizarin Red S | Sigma-Aldrich | A5533 | |
| Bone growth medium (BGM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin/streptomycin | ||
| Formalin solution neutral buffered 10% | Sigma-Aldrich | HT501128 | |
| Hexadecylpyrdinium (cetylpyridinium) chloride monohydrate | Sigma-Aldrich | C9002 | |
| L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate | Sigma-Aldrich | A8960 | |
| MEM alpha medium (α-MEM) | Gibco Life Technologies | 11900-073 | |
| Osteogenic mineralization medium (MM) | α-MEM + 10% heat-inactivated FBS + 1% penicillin-streptomycin + 50 µg/mL ascorbic acid + 5 mM β-glycerophosphate | ||
| Penicillin-streptomycin | Sigma-Aldrich | P4333 | |
| Phosphate-buffered saline 1x (PBS) pH 7.4 | Gibco, Thermo Fisher Scientific | 10010023 | |
| β-Glycerophosphate disodium salt hydrate | Sigma-Aldrich | G5422 | |
| Ultrapure water: Cell culture water pyrogen free | VWR | L0970-500 | |
| 96-well transparent plate | Greiner bio-one | 655001 | |
| 24-well suspension culture plate | Greiner bio-one | 662102 | |
| 24-well tissue culture plate | Greiner bio-one | 662160 | |
| Centrifuge: VWR Mega Star 600R | VWR | ||
| CO2-Incubator: HeraCell 240 | Thermo Scientific | ||
| Flatbed scanner: Epson Perfection 1200 Photo | Epson | ||
| Infinite M200 Pro microplate reader | Tecan | ||
| Microcentrifuge tube: Eppendorf tube safe-lock 1.5 mL | VWR | 21008-959 | |
| Shaking incubator GFL3032 | GFL | 3032 | |
| Single-use pipet: serum pipet | Greiner bio-one | 612301 |
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Cite This Article
In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction. J. Vis. Exp. (Pending Publication), e21167, doi: (2023).